Novel tunable photoactivatable silicon rhodamine fluorophores

ABSTRACT

The invention relates to a compound characterized by general formula (100), wherein R1 and R6 are H or F, R2, R3, R4 and R5 can be any substituent, R7, R8, RN1, RN2, RN3 and RN4 are a hydrocarbon moiety, one of R9 and R10 is hydrogen and the other one is hydrogen or a saturated carbon atom connected to any substituent, and its use in staining and live cell fluorescence imaging.

The present invention relates to a new class of photoactivatable Siliconrhodamine (PA-SiR) fluorophores.

BACKGROUND

Small molecule organic dyes are generally brighter and therefore bettersuited for single-molecule localisation microscopy (SMLM) thanfluorescent proteins, and hence interest in intracellular labellingtechniques using synthetic dyes has grown with the advent ofsuper-resolution microscopy. Photoactivatable synthetic dyes areparticularly powerful tools for studying cellular processes with hightemporal and spatial resolution.

An application of particular interest is the adaption of small moleculedyes to their use with self-labelling protein tags or other proteinlabelling techniques such as click chemistry with unnatural amino acids,enabling their localization to specific cellular targets.

Rhodamines and their silicon derivatives (SiR) are important classes offluorophores used in live cell imaging due to their high brightness(high extinction coefficient (ε) and high quantum yield (ϕ)), theirphotostability, and their cell permeability. WO2013029650A1(PCT/EP2011/064750) discusses the present inventors' previouscontributions to this field. Photoactivatable ortho-nitrobenzylanalogues can be obtained, but were mostly used in fixed cellmicroscopy. When applied in live cells, the stoichiometric formation ofthe coloured and toxic byproduct nitroso-aldehyde is a majordisadvantage. In addition, the o-nitrobenzyl group is bulky and addslipophilicity to the molecule, making the probe less cell permeable thanits non-activatable analogue.

Based on the above-mentioned state of the art, the objective of thepresent invention is to provide a new class of photoactivatablefluorophores with improved properties. This objective is attained by thesubject matter of the below presented claims.

SUMMARY OF THE INVENTION

Herein the inventors disclose that in silicon rhodamines the replacementof an aromatic ring by an alkyl chain makes it possible to obtainnon-fluorescent, but photoactivatable olefinic silicon rhodaminederivatives with an exocyclic double bond. Photoactivation leads to theprotonation of the double bond, which transforms the molecule into acationic fluorescent molecule. Once photoactivated, the cation is inequilibrium with corresponding adducts, formed by inter- orintramolecular nucleophilic attack (e.g. by water resulting inhydroxylated derivatives), similarly to the equilibrium between the openand closed (=spiro-lactone) forms of the SiR fluorophore. Throughchemical modifications or attachment to biomolecules the inventors wereable to fine-tune equilibrium and kinetics of exchange between thecationic fluorescent and neutral non-fluorescent adducts.

TERMS AND DEFINITIONS

The term C₁-C₄ alkyl in the context of the present specificationsignifies a saturated linear or branched hydrocarbon having 1, 2, 3 or 4carbon atoms, wherein in certain embodiments one carbon-carbon bond maybe unsaturated and one CH₂ moiety may be exchanged for oxygen (etherbridge) or nitrogen (NH, or NR with R being methyl, ethyl, or propyl;amino bridge). Non-limiting examples for a C₁-C₄ alkyl are methyl,ethyl, propyl, prop-2-enyl, n-butyl, 2-methylpropyl, tert-butyl,but-3-enyl, prop-2-inyl and but-3-inyl. In certain embodiments, a C₁-C₄alkyl is a methyl, ethyl, propyl or butyl moiety.

A C₁-C₆ alkyl in the context of the present specification signifies asaturated linear or branched hydrocarbon having 1, 2, 3, 4, 5 or 6carbon atoms, wherein one carbon-carbon bond may be unsaturated and oneCH₂ moiety may be exchanged for oxygen (ether bridge) or nitrogen (NH,or NR with R being methyl, ethyl, or propyl; amino bridge). Non-limitingexamples for a C₁-C₆ alkyl include the examples given for C₁-C₄ alkylabove, and additionally 3-methylbut-2-enyl, 2-methylbut-3-enyl,3-methylbut-3-enyl, n-pentyl, 2-methylbutyl, 3-methylbutyl,1,1-dimethylpropyl, 1,2-dimethylpropyl, 1,2-dimethylpropyl, pent-4-inyl,3-methyl-2-pentyl, and 4-methyl-2-pentyl. In certain embodiments, a C₅alkyl is a pentyl or cyclopentyl moiety and a C₆ alkyl is a hexyl orcyclohexyl moiety.

The term unsubstituted C_(n) alkyl when used herein in the narrowestsense relates to the moiety —C_(n)H_(2n)— if used as a bridge betweenmoieties of the molecule, or —C_(n)H_(2n+1) if used in the context of aterminal moiety. It may still contain fewer H atoms if a cyclicalstructure or one or more (non-aromatic) double bonds are present.

The term C_(n) alkylene in the context of the present specificationsignifies a saturated linear or branched hydrocarbon comprising one ormore double bonds. An unsubstituted alkylene consists of C and H only. Asubstituted alkylene may comprise one or several substituents as definedherein for substituted alkyl.

The term C_(n) alkylyne in the context of the present specificationsignifies a saturated linear or branched hydrocarbon comprising one ormore triple bonds and may also comprise one or more double bonds inaddition to the triple bond(s). An unsubstituted alkylyne consists of Cand H only. A substituted alkylyne may comprise one or severalsubstituents as defined herein for substituted alkyl.

The terms unsubstituted C_(n) alkyl and substituted C_(n) alkyl includea linear alkyl comprising or being linked to a cyclical structure, forexample a cyclopropane, cyclobutane, cyclopentane or cyclohexane moiety,unsubstituted or substituted depending on the annotation or the contextof mention, having linear alkyl substitutions. The total number ofcarbon and—where appropriate—N, O or other hetero atoms in the linearchain or cyclical structure adds up to n.

Where used in the context of chemical formulae, the followingabbreviations may be used: Me is methyl CH₃, Et is ethyl —CH₂CH₃, Propis propyl —(CH₂)₂CH₃ (n-propyl, n-pr) or —CH(CH₃)₂ (iso-propyl, i-pr),but is butyl —C₄H₉, —(CH₂)₃CH₃, —CHCH₃CH₂CH₃, —CH₂CH(CH₃)₂ or —C(CH₃)₃.

The term substituted alkyl in its broadest sense refers to an alkyl asdefined above in the broadest sense that is covalently linked to an atomthat is not carbon or hydrogen, particularly to an atom selected from N,O, F, B, Si, P, S, Se, CI, Br and I, which itself may be—ifapplicable—linked to one or several other atoms of this group, or tohydrogen, or to an unsaturated or saturated hydrocarbon (alkyl or arylin their broadest sense). In a narrower sense, substituted alkyl refersto an alkyl as defined above in the broadest sense that is substitutedin one or several carbon atoms by groups selected from amine NH₂,alkylamine NHR, imide NH, alkylimide NR, amino(carboxyalkyl) NHCOR orNRCOR, hydroxyl OH, oxyalkyl OR, oxy(carboxyalkyl) OCOR, carbonyl O andits ketal or acetal (OR)₂, nitril CN, isonitril NC, cyanate CNO,isocyanate NCO, thiocyanate CNS, isothiocyanate NCS, fluoride F, chorideCl, bromide Br, iodide I, phosphonate PO₃H₂, PO₃R₂, phosphate OPO₃H₂ andOPO₃R₂, sulfhydryl SH, suflalkyl SR, sulfoxide SOR, sulfonyl SO₂R,sulfanylamide SO₂NHR, sulfate SO₃H and sulfate ester SO₃R, wherein the Rsubstituent as used in the current paragraph, different from other usesassigned to R in the body of the specification, is itself anunsubstituted or substituted C₁ to C₁₂ alkyl in its broadest sense, andin a narrower sense, R is methyl, ethyl or propyl unless otherwisespecified.

It is understood that mention of moieties SO₃H or COOH or other acidicgroups imply presence of the deprotonated form in the alternative,assuming appropriate conditions that allow dissociation.

The term amino substituted alkyl or hydroxyl substituted alkyl refers toan alkyl according to the above definition that is modified by one orseveral amine or hydroxyl groups NH₂, NHR, NR₂ or OH, wherein the Rsubstituent as used in the current paragraph, different from other usesassigned to R in the body of the specification, is itself anunsubstituted or substituted C₁ to C₁₂ alkyl in its broadest sense, andin a narrower sense, R is methyl, ethyl or propyl unless otherwisespecified. An alkyl having more than one carbon may comprise more thanone amine or hydroxyl. Unless otherwise specified, the term “substitutedalkyl” refers to alkyl in which each C is only substituted by at mostone amine or hydroxyl group, in addition to bonds to the alkyl chain,terminal methyl, or hydrogen.

The term carboxyl substituted alkyl refers to an alkyl according to theabove definition that is modified by one or several carboxyl groupsCOOH, or derivatives thereof, particularly carboxylamides CONH₂, CONHRand CONR₂, or carboxylic esters COOR, with R having the meaning as laidout in the preceding paragraph and different from other meaningsassigned to R in the body of this specification.

Non-limiting examples of amino-substituted alkyl include —CH₂NH₂,—CH₂NHMe, —CH₂NHEt, —CH₂CH₂NH₂, —CH₂CH₂NHMe, —CH₂CH₂NHEt, —(CH₂)₃NH₂,—(CH₂)₃NHMe, —(CH₂)₃NHEt, —CH₂CH(NH₂)CH₃, —CH₂CH(NHMe)CH₃,—CH₂CH(NHEt)CH₃, —(CH₂)₃CH₂NH₂, —(CH₂)₃CH₂NHMe, —(CH₂)₃CH₂NHEt,—CH(CH₂NH₂)CH₂CH₃, —CH(CH₂NHMe)CH₂CH₃, —CH(CH₂NHEt)CH₂CH₃,—CH₂CH(CH₂NH₂)CH₃, —CH₂CH(CH₂NHMe)CH₃, —CH₂CH(CH₂NHEOCH₃,—CH(NH₂)(CH₂)₂NH₂, —CH(NHMe)(CH₂)₂NHMe, —CH(NHEt)(CH₂)₂NHEt,—CH₂CH(NH₂)CH₂NH₂, —CH₂CH(NHMe)CH₂NHMe, —CH₂CH(NHEt)CH₂NHEt,—CH₂CH(NH₂)(CH₂)₂NH₂, —CH₂CH(NHMe)(CH₂)₂NHMe, —CH₂CH(NHEt)(CH₂)₂NHEt,—CH₂CH(CH₂NH₂)₂, —CH₂CH(CH₂NHMe)₂ and —CH₂CH(CH₂NHEt)₂ for terminalmoieties and —CH₂CHNH₂—, —CH₂CHNHMe-, —CH₂CHNHEt- for an aminosubstituted alkyl moiety bridging two other moieties.

Non-limiting examples of hydroxy-substituted alkyl include —CH₂OH,—(CH₂)₂OH, —(CH₂)₃OH, —CH₂CH(OH)CH₃, —(CH₂)₄OH, —CH(CH₂OH)CH₂CH₃,—CH₂CH(CH₂OH)CH₃, —CH(OH)(CH₂)₂OH, —CH₂CH(OH)CH₂OH, —CH₂CH(OH)(CH₂)₂OHand —CH₂CH(CH₂OH)₂ for terminal moieties and —CHOH—, —CH₂CHOH—,—CH₂CH(OH)CH₂—, —(CH₂)₂CHOHCH₂—, —CH(CH₂OH)CH₂CH₂—, —CH₂CH(CH₂OH)CH₂—,—CH(OH)(CH₂CHOH—, —CH₂CH(OH)CH₂OH, —CH₂CH(OH)(CH₂)₂OH and—CH₂CHCH₂OHCHOH— for a hydroxyl substituted alkyl moiety bridging twoother moieties.

The term halogen-substituted alkyl refers to an alkyl according to theabove definition that is modified by one or several halogen atomsselected (independently) from F, Cl, Br, I.

The term fluoro substituted alkyl refers to an alkyl according to theabove definition that is modified by one or several fluoride groups F.Non-limiting examples of fluoro-substituted alkyl include —CH₂F, —CHF₂,—CF₃, —(CH₂)₂F, —(CHF)₂H, —(CHF)₂F, —C₂F₅, —(CH₂)₃F, —(CHF)₃H, —(CHF)₃F,—C₃F₇, —(CH₂)₄F, —(CHF)₄H, —(CHF)₄F and —C₄F₉.

Non-limiting examples of hydroxyl- and fluoro-substituted alkyl include—CHFCH₂OH, —CF₂CH₂OH, —(CHF)₂CH₂OH, —(CF₂)₂CH₂OH, —(CHF)₃CH₂OH,—(CF₂)₃CH₂OH, —(CH₂)₃OH, ——CF₂CH(OH)CH₃, —CF₂CH(OH)CF₃,—CF(CH₂OH)CHFCH₃, and —CF(CH₂OH)CHFCF₃.

The term aryl in the context of the present invention signifies a cyclicaromatic C₅-C₁₀ hydrocarbon that may comprise a heteroatom (e.g. N, O,S). Examples of aryl include, without being restricted to, phenyl andnaphthyl, and any heteroaryl. A heteroaryl is an aryl that comprises oneor several nitrogen, oxygen and/or sulphur atoms. Examples forheteroaryl include, without being restricted to, pyrrole, thiophene,furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine,thiazin, quinoline, benzofuran and indole. An aryl or a heteroaryl inthe context of the invention additionally may be substituted by one ormore alkyl groups.

An aryl methylene in the context of the present invention signifies aCH₂ (-methylene) group substituted by an aryl moiety. One non-limitingexample of aryl methylene is a benzyl (Bn) group. If used in particular,a heteroaryl methylene in the context of the present invention signifiesa CH₂ (-methylene) group substituted by a heteroaryl moiety.

A substituted aryl or heteroaryl or aryl methylene may comprise one orseveral substituents as defined herein for substituted alkyl.

“Capable of forming a hybrid” in the context of the present inventionrelates to sequences that under the conditions existing within thecytosol of a mammalian cell, are able to bind selectively to theirtarget sequence. Such hybridizing sequences may be contiguouslyreverse-complimentary to the target sequence, or may comprise gaps,mismatches or additional non-matching nucleotides. The minimal lengthfor a sequence to be capable of forming a hybrid depends on itscomposition, with C or G nucleotides contributing more to the energy ofbinding than A or T/U nucleotides, and the backbone chemistry.

“Nucleotides” in the context of the present invention are nucleic acidor nucleic acid analogue building blocks, oligomers of which are capableof forming selective hybrids with RNA oligomers on the basis of basepairing. The term nucleotides in this context includes the classicribonucleotide building blocks adenosine, guanosine, uridine (andribosylthymin), cytidine, the classic deoxyribonucleotidesdeoxyadenosine, deoxyguanosine, thymidine, deoxyuridine anddeoxycytidine. It further includes analogues of nucleic acids such asphosphotioates, 2′O-methylphosphothioates, peptide nucleic acids (PNA;N-(2-aminoethyl)-glycine units linked by peptide linkage, with thenucleobase attached to the alpha-carbon of the glycine) or lockednucleic acids (LNA; 2′O, 4′C methylene bridged RNA building blocks). Thehybridizing sequence may be composed of any of the above nucleotides, ormixtures thereof.

In the following, the molecules of the invention are discussed indetail. Where ionizable moieties are disclosed, it is understood thatany salt, particularly any pharmaceutically acceptable salt of suchmolecule is encompassed by the invention. The salt comprises the ionizedmolecule of the invention and an oppositely charged counterion.Non-limiting examples of anionic salt forms include acetate, benzoate,besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate,edisylate, embonate, estolate, fumarate, gluceptate, gluconate,hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate,maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate,napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate,disalicylate, stearate, succinate, sulfate, tartrate, tosylate,triethiodide and valerate. Non-limiting examples of cationic salt formsinclude aluminium, benzathine, calcium, ethylene diamine, lysine,magnesium, meglumine, potassium, procaine, sodium, tromethamine andzinc.

It is understood that any position wherein H is present can besubstituted by D.

A first aspect of the invention relates to a compound characterized bygeneral formula (100):

R¹ and R⁶ are independently selected from hydrogen and fluorine. Incertain embodiments, R¹ and R⁶ are hydrogen.

R², R³, R⁴ and R⁵ independently of each other can be any substituent.The skilled person is aware that these positions are highly variable inthe art of rhodamine chemistry. If the molecule of the invention is tobe connected to, for example, a tag or selective targeting moiety, thesepositions (in addition to one of R⁹ and R¹⁰) offer themselves to thisfunction, as derivatization by an alkyl linker will not greatly affectoptical properties.

In certain embodiments, R², R³, R⁴ and R⁵ independently of each otherare selected from H and a moiety having a molecular weight between 15and 250 u (g/mol).

In certain embodiments, R², R³, R⁴ and R⁵ independently of each otherare selected from H, halogen, SO₃H, CO₂H, NO₂, CO₂R, SO₂R (with R beingselected from C₁ to C₄ unsubstituted alkyl) and an unsubstituted orsubstituted (particularly unsubstituted or halogen-, amino-, hydroxyl-,SO₃H— and/or carboxyl substituted) moiety selected from C₁-C₂₀ alkyl,C₃-C₈ cycloalkyl, C₁-C₂₀ alkoxy, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, C₇-C₂₀alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combinationthereof.

R⁷ and R⁸ are independently selected from unsubstituted and substituted,particularly amino-, hydroxy-, halogen-, SO₃ ⁻— and/orcarboxy-substituted, C₁-C₁₂ alkyl, C₃-C₈ cycloalkyl, C₂-C₁₂ alkylene,C₂-C₁₂ alkylyne and C₇-C₁₂ alkylaryl and unsubstituted or substituted5 - or 6-ring aryl.

In certain embodiments, R⁷ and R⁸ are selected from an substituted,particularly amino-, hydroxy-, halogen-, SO₃ ⁻— and/orcarboxy-substituted, or unsubstituted alkyl, alkenyl, alkenyl, alkylaryor aryl, and the total number of carbon atoms in both moieties does notexceed 12. In certain embodiments, this total carbon number does notexceed 10, 8 or even six.

In certain embodiments, R⁷ and R⁸ are the same and each comprises 1, 2,3, 4, 5 or 6 carbon atoms. In certain embodiments, R⁷ and R⁸ are bothmethyl, ethyl, isopropyl, n-propyl, n-butyl, isobutyl or tert-butyl.

In certain embodiments, R⁷ and R⁸ are both phenyl or benzyl.

The N substituents R^(N1), R^(N2), R^(N3) and R^(N4) can be variedwidely and allow tuning of the optical properties of the compound. Thecompound may also be attached to a tag or targeting moiety through thesepositions.

R^(N1), R^(N2), R^(N3) and R^(N4) may be independently selected from H,unsubstituted and substituted, particularly amino-, hydroxy-, halogen-,SO₃ ⁻— and/or carboxy-substituted, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₁-C₄acyl (particularly acetyl), and C₇-C₁₂ alkylaryl (particularly methylenearyl, more particularly benzyl), and unsubstituted phenyl or phenylsubstituted by COOH—, COOR, CONR₂, unsubstituted alkyl, halogen,O-alkyl, and/or NO₂.

Alternatively, R^(N1) together with R^(N2), and/or R^(N3) together withR^(N4) are a C₃, C₄, C₆ unsubstituted or substituted, particularlyamino-, hydroxy-, halogen-, SO₃ ⁻— and/or carboxy-substituted, alkylforming a 3-7 sized ring structure. For illustration, if the twosubstituents on N together are a C₃ unsubstituted alkyl, the resultingstructure is an azetidine (4-membered ring). The ring structure thusformed may comprise heteroatoms (to form a morpholine, for example), andmay be substituted, particularly by short unsubstituted alkyl, (e.g.methyl).

Alternatively, R^(N1) and/or R^(N3) are independently selected from Hand unsubstituted and substituted, particularly amino-, hydroxy-,halogen-, SO₃ ⁻— and/or carboxy-substituted, C₁-C₈ alkyl, C₃-C₈cycloalkyl, C₁-C₄ acyl, and C₇-C₁₂ alkylaryl, and R^(N2) together withR² or R³, and/or R^(N4) together with R⁴ or R⁵, is an unsubstituted orsubstituted C₂, C₃ or C₄ alkyl, or an unsubstituted or substituted C₂,C₃ or C₄ N-, O-, S-, or Se-alkyl forming a ring structure. It is alsopossible that the N substituents form two ring structures with R² andR³, to either of which ring structures the limitations given in thisparagraph may apply. Such ring structures are well known in rhodaminechemistry.

In embodiments where only substituents of one N participate in ring, theother N's substituents are selected as laid out in previous paragraph.

One of R⁹ and R¹⁰ is hydrogen and the other one is hydrogen or asaturated carbon atom connected to any substituent. In certainembodiments, the moiety connected to the saturated carbon atom isselected from H and a moiety having a molecular weight between 15 and250 u (g/mol). In certain embodiments, the moiety connected to thesaturated carbon atom is selected from H, halogen, SO₃H, CO₂H, NO₂,CO₂R, SO₂R (with R being selected from C₁ to C₄ unsubstituted alkyl) andan unsubstituted or substituted (particularly unsubstituted or halogen-,amino-, hydroxyl-, SO₃H— and/or carboxyl substituted) moiety selectedfrom C₁-C₂₀ alkyl, C₃-C₈ cycloalkyl, C₁-C₂₀ alkoxy, C₂-C₂₀ alkenyl,C₂-C₂₀ alkynyl, C₇-C₂₀ alkylaryl, phenyl and 5- or 6-membered ringheteroaryl, or a combination thereof. In particular embodiments, thenon-H moiety is an alkyl, alkenyl, or linker moiety connecting thefluorophore system to a binding moiety M.

In certain embodiments, R², R³, R⁴ and R⁵ are independently selectedfrom halogen, H, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl),amine, NO₂, CHO, COOH, C(O)NR^(N5) ₂, COO(alkyl), COO(aryl), PO₃H₂,SO₃H, and C₁ to C₄ alkyl, alkyl being optionally substituted with one ormore heteroatoms independently selected from N, O, and S, halogen, OH,O(alkyl), O(aryl), C(O)NR⁵ ₂, SH, S(alkyl), S(aryl), amine, NO₂, CHO,COO⁻, COOH, and with R^(N5) selected from H and unsubstituted or amino-,hydroxyl-, carboxyl or fluoro substituted C₁ to C₆ alkyl, particularlyR^(N5) is selected from H and unsubstituted C₁ to C₃ alkyl.

In certain embodiments, the compound of the invention is covalentlylinked to a binding moiety M via any of the substituents R¹, R², R³, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^(N1), R^(N2), R^(N3) or R^(N4).

In certain embodiments, the compound is covalently linked (particularlythrough any one of substituents R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R^(N1), R^(N2), R^(N3) and R^(N4)) to a binding moiety M. M is atargeting or tagging moiety that allows the core dye compound to beselectively attached to biomolecules or certain target structuresselected from:

M may be a moiety selectively attachable by covalent bond to a proteinor nucleic acid under conditions prevailing in cell culture or inside ofa living cell. In certain embodiments, M is a moiety able to form anester bond, an ether bond, an amide bond, a disulfide bond, a Schiff'base, or react in a click-chemistry reaction. In certain embodiments, Mis selected from —COCH═CH₂, allowing Michael addition of SH groupspresent in proteins, —CO—NHS (N-hydroxysuccinimide), biotin, and anazide or ethyne moiety (click cycloaddition), tetrazine or BCN(bicyclo[6.1.0]nonyne), SCO (Cyclooctyne) or TCO (Transcyclooctene),maleimide to react with SH groups. The skilled artisan is aware ofvarious alternatives of click chemistry partners that may alternativelybe employed.

M may alternatively be a moiety a substrate ofO⁶-alkylguanine-DNA-alkyltransferase, particularly a6-[(4-methylenephenyl)methoxy]-9H-purin-2-amine moiety of formula (110),or a pyrimidine derivative thereof, particularly a moiety of formula(111) or (112),

or a substrate of a haloalkane halotransferase, particularly a1-chlorohexyl moiety as exemplarily shown below;

M may also be a substrate of dihydrofolate reductase DHFR, particularlythe moiety:

If M is selected to function as a targeting moiety for biomolecules, aparticularly advantageous approach is to covalently link the dye to adrug or drug-like small molecule with high specificity for a particularprotein, nucleic acid or other complex biological structure. Thus, incertain embodiments, M is a moiety capable of selectively interactingnon-covalently with a biomolecule (particularly a protein or nucleicacid) under inside of a live cell, wherein said moiety and saidbiomolecule form a complex having a dissociation constant kip of 10⁻⁶mol/1 or less. In certain such embodiments, M has a molecular mass ofmore than 160 u but less than 1000 u, particularly less than 700 u, moreparticularly less than 500 u, and M comprises up to five hydrogen bonddonators (e.g., oxygen and or nitrogen atoms with one H attached), up toten hydrogen bond acceptors (e.g., oxygen or nitrogen atoms) and ischaracterized by an octanol-water partition coefficient logP of below5.6 (any of these characteristics applied to the isolated M moiety,without regard to the rest of compound). These are the so-called“Lipinski” rules of 5 (originally, referring to molecules between 160and 500 u) for drug-like compounds. M can be any licensed medicinal orveterinary drug or a drug candidate for which affinity data for aparticular biomolecule are known.

In certain particular embodiments, M is selected from taxol (see e.g.compound 56), jasplaklinolide (see e.g. compound 49), a bis-benzimideDNA stain (Hoechst dyes 33342, 33258, 34580; see compd 51), pepstatin A(specific staining of lysosomes), and triphenylphosphonium (specificstaining of mitochondria).

In certain particular embodiments, M is an oligonucleotide having asequence length of 10 to 40 nucleotides capable of sequence specificallyforming a hybrid with a DNA or RNA sequence, particularly inside ahuman, animal, plant or bacterial cell.

In certain particular embodiments, M is a lipid. In certain embodiments,M is a lipid selected from a ceramide derivative, a glyceride, or afatty acid.

In certain embodiments, any one of substituents R², R³, R⁴, R⁵, and oneof R⁹ and R¹⁰ independently of any other is H or a moiety having amolecular weight between 15 and 1500 u (g/mol) constituted of C, H, D,N, O, Si, P, S, Se F, CI, Br, I atoms.

In certain particular embodiments, one of substituents R², R³, R⁴, R⁵,R⁹ and R¹⁰ is a moiety having a molecular weight between 15 and 1500 uand the other ones are selected from H and unsubstituted or fluoro-,amino-, hydroxyl-, SO₃H— and/or carboxyl substituted C₁ to C₄ alkyl,alkenyl or alkynyl.

In certain particular embodiments, one of substituents R², R³, R⁴, R⁵,R⁹ and R¹⁰ is a moiety having a molecular weight between 15 and 1500 uand the other ones are H.

In certain embodiments, the moiety having a molecular weight between 15and 1500 u is characterized by a general formula —L-M, wherein L is alinker covalently connecting the compound of structure (1) to thebinding moiety M as defined above, and L is a covalent bond or a linkerconsisting of 1 to 50 atoms having an atomic weight of 12 or higher (andH to fill valencies).

In general, the gist of the invention encompasses the attachment of aspecific tagging or targeting moiety M to the central fluorophore, andthe skilled artisan is aware that a very large number of alternativesexist to link the central fluorophore to M. In certain particularembodiments, the moiety having a molecular weight between 15 and 1500 uis characterized by a general formula

-L^(A1) _(n)-L^(J1) _(n)′-L^(A2) _(m)-L^(J2) _(m)′-L^(A3) _(p)-L^(j3)_(p)′-L^(A4) _(q)-L^(J4) _(q)′-M_(s), wherein

-   -   L^(A1), L^(A2), L^(A3) and L^(A4) independently of each other        are selected from C₁ to C₁₂ unsubstituted or amino-, hydroxyl-,        carboxyl- or fluoro substituted alkyl or cycloalkyl,        (CH₂—CH₂—O)_(r) or (CH₂—CH(OH)—CH₂—O)_(r) with r being an        integer from 1 to 20, alkylaryl (e.g. benzyl), alkylaryl-alkyl        (e.g. CH₂-phenyl-CH₂), and unsubstituted or alkyl-, halogen-,        amino-, alkylamino-, imido-, nitro-, hydroxyl-oxyalkyl-,        carbonyl-, carboxyl-, sulfuryl- and/or sulfoxyl substituted aryl        or heteroaryl,    -   L^(J1), L_(J2), L^(J3) and L^(J4) independently of each other        are selected from —NR⁵C(O)—, —C(O)N(R⁵)— (amide), —CN—, —NC—        (Schiff base), —CO—, —OC(O)—, —C(O)O— (ester), —NR^(N5)—, —O—,        —P(OOH)—, —OP(OOH)—, —P(OOH)O—, —OP(OOH)O—, —OP(OOH)O—, —S—,        —SO—, SO₂—,        -   with R^(N5) selected from H and unsubstituted or amino-,            hydroxyl-, carboxyl or fluoro substituted C₁ to C₆ alkyl,            particularly R^(N5) is selected from H and unsubstituted C₁            to C₃ alkyl;    -   n, n′, m, m′, p, p′, q, q′ and s independently from each other        are selected from 0 and 1,    -   and    -   M has the meaning defined above.

In certain particular embodiments, L is -L^(A1)-L^(J1)-L^(A2)_(m)-L^(J2) _(m)′-L^(A3) _(p), wherein

-   -   -L^(A1), L^(A2) and L^(A3) are independently selected from C₁ to        C₆ unsubstituted, amino-, hydroxyl-, carboxyl- or fluoro        substituted alkyl or cycloalkyl, and (CH₂—CH₂—O), or        (CH₂—CH(OH)—CH₂—O)_(r) with r being an integer from 1 to 4, and    -   L^(J1) and L^(J2) are selected independently from —NR⁵C(O)—,        —C(O)N(R⁵)—, —CN—, —NC—, —CO—, —OC(O)—, —C(O)O—, NR^(N5)—, —O—,        and —S—, and    -   m, m′ and p independently from each other are selected from 0        and 1.

In certain embodiments, R⁷ and R⁸ are independently selected from

-   -   unsubstituted or hydroxyl-, amino- or halogen-substituted C₁ to        C₄ alkyl, alkenyl or alkynyl,    -   unsubstituted or hydroxyl-, amino- or halogen-substituted C₃ to        C₆ cycloalkyl or    -   unsubstituted or hydroxyl-, alkyoxy-, amino- or        halogen-substituted phenyl.

In certain particular embodiments, R⁷ and R⁸ are methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, or phenyl. In evenmore particular embodiments, R⁷ and R⁸ are the same.

In certain embodiments, the N substituents on one or both N are selectedindividually and do not form a ring structure. Herein, R^(N1) andR^(N2), and/or R^(N3) and R^(N4), are independently selected from H,unsubstituted and amino-, hydroxy-, carboxy- and/or fluoro-substitutedC₁-C₆ alkyl, C₁-C₄ acyl, and C₃-C₆ cycloalkyl.

In certain particular embodiments, R^(N1) and R^(N2), and/or R^(N3) andR^(N4), are independently selected from H, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl and CH₂CF₃.

In certain embodiments, the N substituents on one or both N are selectedtogether as one linear chain to form a ring structure. Herein, R^(N1)together with R^(N2), and/or R^(N3) together with R^(N4) together are anunsubstituted or alkyl-, amino-, hydroxy-, carboxy- and/orfluoro-substituted C₃-C₆ alkyl, particularly —(CH₂)₃—, —(CH₂)₄—,—(CH₂)₅—, —(CH₂)₂O(CH₂)₂— or —(CH₂)₂NR^(NN)(CH₂)₂— with R^(NN) beingselected from H and unsubstituted C₁ to C₄ alkyl. In certainembodiments, one or several of the ring carbon atoms may be substitutedby methyl.

In certain embodiments, the N substituents on one or both N are selectedto form a ring structure bridging the N and positions R²/R⁵ and/or R³/R⁴on the dibenzo[b,e]silin-10(5H)-ylidene ring core.

If one ring connects N and R²/R⁵, R^(N1) and/or R^(N3) are independentlyselected from H, unsubstituted and alkyl- (particularly methyl-),amino-, hydroxy-, carboxy- and/or fluoro-substituted C₁-C₆ alkyl, C₁-C₄acyl, and C₃-C₆ cycloalkyl, and R^(N2) together with R² or R³, and/orR^(N4) together with R⁴ or R⁵, is an alkyl or heteroalkyl bridgeselected from —(CH₂)₂—, —(CH₂)₃—, —CH₂CH═CH— or —(CH₂)₄— or —CH₂—O—,—CH₂—NR⁵—, —CH₂—S—, —CH₂—Se—, —(CH₂)₂O—, —(CH₂)₂NR^(N)—, —(CH₂)₂S—,—(CH₂)₂Se—, —CH₂—O—CH₂—, —CH₂NR⁵—, —CH₂S—CH₂—, —CH₂—Se—CH₂—,—CH₂-(1,2)phenyl-, and a mono- or dimethyl substituted derivative of anyone of the foregoing alkyl or heteroalkyl bridge moieties. It isunderstood that when only one ring structure is formed between N andposition R²/R⁵, the N not participating in the ring structure can besubstituted by any of the options laid out above, namely independent orsame alkyl or aryl, or one alkyl forming a four to seven membered ring.

In certain particular embodiments, R^(N1) and/or R^(N3) areindependently selected from H, unsubstituted and alkyl- (particularlymethyl-), amino-, hydroxy-, carboxy- and/or fluoro-substituted C₁-C₆alkyl, C₁-C₄ acyl, and C₃-C₆ cycloalkyl, and R^(N2) together with R²,and/or R^(N4) together with R⁵, form an annular structure according toany one of substructures (101) to (104). Methods for introducingsubstructures as shown in (101) to (109) and (101′) to (109′) aredisclosed in U.S. Pat. Nos. 6,191,278 and 6,130,101, incorporated hereinby reference.

Herein, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are selected from H,unsubstituted or hydroxyl-, amino-, carboxyl-, sulfoxyl- orhalogen-substituted C₁ to C₄ alkyl, halogen, SO₃R, COOR′, CONR′₂ with R′selected from H and unsubstituted C₁ to C₄ alkyl; R¹⁷—whereapplicable—is selected from H unsubstituted or hydroxyl-, amino-,carboxyl-, sulfoxyl- or halogen-substituted C₁ to C₄ alkyl, halogen,NO₂, CN, SO₃R, COOR′, CONR′₂ with R′ selected from H and unsubstitutedC₁ to C₄ alkyl; and R¹, R³, R⁷ and R³ can have any of the meanings givenelsewhere herein.

In certain particular embodiments of structures (101) to (104), R¹¹,R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are selected from H, methyl, CH₂—SO₃H, Cl andF.

Alternatively, R^(N1) together with R³, and R^(N2) together with R²,and/or R^(N3) together with R⁴, and R^(N4) together with R⁵, form abi-annular structure according to any one of substructures (105) to(107) and/or (105′) to (107′):

wherein R¹¹, R¹², R¹³, and R¹⁵ are selected from H, unsubstituted orhydroxyl-, amino-, carboxyl-, sulfoxyl- or halogen-substituted C₁ to C₄alkyl, halogen, SO₃R, COOR′, CONR′₂ with R selected from H andunsubstituted C₁ to C₄ alkyl; and R¹, R³, R⁷ and R⁸ can have any of themeanings given elsewhere herein.

In certain particular embodiments of structures (105) to (107), R¹¹,R¹², R¹³, and R¹⁵ are selected from H, methyl, CH₂—SO₃H, Cl and F.

Alternatively, R^(N2) and/or R^(N4) are independently selected from H,unsubstituted and alkyl-(particularly methyl-), amino-, hydroxy-,carboxy- and/or fluoro-substituted C₁-C₆ alkyl, C₁-C₄ acyl, and C₃-C₆cycloalkyl, and R^(N1) together with R³, and/or R^(N3) together with R⁴,form an annular structure according to any one of substructures (108) to(109) and/or (108′) to (109′):

wherein R¹, R³, R⁷ and R⁸ can have any of the meanings given herein.

In certain particular embodiments, the N substituents together form afour-membered ring (azetidine). Herein, R^(N1) together with R^(N2),and/or R^(N3) together with R^(N4) together are selected from —(CH₂)₃—,—CH₂CHFCH₂—, —CH₂CF₂CH₂—, —CH₂CH(CH₃)CH₂—, —CH₂C(CH₃)₂CH₂—,CH₂CH(CN)CH₂—, CH₂CH(COOH)CH₂—, CH₂CH(CH₂COOH)CH₂—, —CH₂CH(OCH₃)CH₂— and—CH₂CH(N(CH₃)₂)CH₂—. In certain more particular embodiments thereof, thesubstituent is the same for R^(N1) with R^(N2), and R^(N3) with R^(N4).

US2017045501, incorporated herein in by reference, discloses methods formaking the above azetidine rings.

In certain particular embodiments, R¹, R⁶ and R⁹ are H.

In certain particular embodiments, R² and R⁵ are F or Cl.

In certain particular embodiments, R¹, R⁶ and R⁹ are H and R² and R⁵ areF or Cl.

In certain particular embodiments, R², R³, R⁴ and R⁵ are selected fromH, halogen, SO₃H, and unsubstituted and amino-, hydroxy-, carboxy-,SO₃H—, and/or halogen-substituted C₁-C₄ alkyl, CO₂H, CO₂R, SO₂R with Rbeing selected from C₁ to C₄ unsubstituted alkyl.

In certain particular embodiments, R⁷ and R⁸ are independently selectedfrom unsubstituted, or halogen-substituted C₁ to C₄ alkyl or C₃ to C₆cycloalkyl and phenyl.

In certain particular embodiments, R^(N1), R^(N2), R^(N3) and R^(N4) areindividually unsubstituted or amino-, hydroxyl- or halogen-substitutedC₁ to C₄ alkyl or C₃ to C₆ cycloalkyl, or R^(N1) together with R^(N2),and R^(N3) together with R^(N4) together with the N form anunsubstituted or methyl-, ethyl- propyl-, or halogen-substitutedaziridine, pyrrolidine, piperidine, piperazine or morpholine.

In certain particular embodiments, R¹⁹ is selected from unsubstituted oramino-, hydroxyl-, carboxyl- and/or halogen-substituted C₂ to C₁₂ alkylor C₃ to C₇ cycloalkyl.

In certain particular embodiments, R¹⁰ is -L^(A1) _(n)-L^(J1)_(n)′-L^(A2) _(m)-L^(J2) _(m)′-L^(A3) _(p)-L^(J3) _(p)′-L^(A4)_(q)-L^(J4) _(q)′-M_(s), wherein L^(A1 . . . 4), L^(J1 . . . 4), n, n′ .. . q′, s and M have the definitions recited above.

Certain particular embodiments relate to the following selection offeatures, with all undefined features being selectable from otherparticular embodiments or general descriptions of the missing features:

-   -   R¹, R⁶ and R⁹ are H, and    -   R², R³, R⁴ and R⁵ are selected from H, halogen, SO₃H, and        unsubstituted and amino-, hydroxy-, SO₃H—, carboxy- and/or        halogen-substituted C₁-C₄ alkyl, CO₂H, CO₂R, SO₂R with R being        selected from C₁ to C₄ unsubstituted alkyl.

Another particular combination is:

-   -   R¹, R⁶ and R⁹ are H, and    -   R⁷ and R⁹ are independently selected from unsubstituted or        halogen-substituted C₁ to C₄ alkyl or C₃ to C₆ cycloalkyl and        phenyl.

Another particular combination is:

-   -   R¹, R⁶ and R⁹ are H, and    -   R², R³, R⁴ and R⁵ are selected from H, halogen, SO₃H, and        unsubstituted and amino-, hydroxy-, SO₃H—, carboxy- and/or        halogen-substituted C₁-C₄ alkyl, CO₂H, CO₂R, SO₂R with R being        selected from C₁ to C₄ unsubstituted alkyl, and    -   R⁷ and R⁸ are independently selected from unsubstituted or        halogen-substituted C₁ to C₄ alkyl or C₃ to C₆ cycloalkyl and        phenyl.

Another particular combination is:

-   -   R¹, R⁶ and R⁹ are H, and    -   R⁷ and R⁹ are independently selected from unsubstituted or        halogen-substituted C₁ to C₄ alkyl or C₃ to C₆ cycloalkyl and        phenyl, and    -   R^(N1), R^(N2), R^(N3) and R^(N4) are individually unsubstituted        or amino-, hydroxyl- or halogen-substituted C₁ to C₄ alkyl or C₃        to C₆ cycloalkyl, or R^(N1) together with R^(N2), and R^(N3)        together with R^(N4) together with the N form an unsubstituted        or methyl-, ethyl- propyl-, or halogen-substituted aziridine,        pyrrolidine, piperidine, piperazine or morpholine and/or

Another particular combination is:

-   -   R¹, R⁶ and R⁹ are H, and    -   R², R³, R⁴ and R⁵ are selected from H, halogen, SO₃H, and        unsubstituted and amino-, hydroxy-, SO₃H—, carboxy- and/or        halogen-substituted C₁-C₄ alkyl, CO₂H, CO₂R, SO₂R with R being        selected from C₁ to C₄ unsubstituted alkyl, and    -   R^(N1), R^(N2), R^(N3) and R^(N4) are individually unsubstituted        or amino-, hydroxyl- or halogen-substituted C₁ to C₄ alkyl or C₃        to C₆ cycloalkyl, or R^(N1) together with R^(N2), and R^(N3)        together with R^(N4) together with the N form an unsubstituted        or methyl-, ethyl- propyl-, or halogen-substituted aziridine,        pyrrolidine, piperidine, piperazine or morpholine.

Another particular combination is:

-   -   R¹, R⁶ and R⁹ are H, and/or    -   R², R³, R⁴ and R⁵ are selected from H, halogen, SO₃H, and        unsubstituted and amino-, hydroxy-, SO₃H—, carboxy- and/or        halogen-substituted C₁-C₄ alkyl, CO₂H, CO₂R, SO₂R with R being        selected from C₁ to C₄ unsubstituted alkyl, and/or    -   R⁷ and R³ are independently selected from unsubstituted or        halogen-substituted C₁ to C₄ alkyl or C₃ to C₆ cycloalkyl and        phenyl, and/or    -   R^(N1), R^(N2), R^(N3) and R^(N4) are individually unsubstituted        or amino-, hydroxyl- or halogen-substituted C₁ to C₄ alkyl or C₃        to C₆ cycloalkyl, or R^(N1) together with R^(N2), and R^(N3)        together with R^(N4) together with the N form an unsubstituted        or methyl-, ethyl- propyl-, or halogen-substituted aziridine,        pyrrolidine, piperidine, piperazine or morpholine and/or    -   R¹⁰ is selected from        -   unsubstituted or amino-, hydroxyl-, carboxyl- and/or            halogen-substituted C₂ to C₁₂ alkyl or C₃ to C₇ cycloalkyl;        -   -L^(A1) _(n)-L^(J1) _(n)′-L^(A2) _(m)-L^(J2) _(m)′-L^(A3)            _(p)-L^(J3) _(p)′-L^(A4) _(q)-L^(J4) _(q)′-M_(s), wherein            L^(A1 . . . 4), L^(J1 . . . 4), n, n′. . . q′, s and M have            the definitions recited above.

Another particular combination is:

-   -   R¹, R², R³, R⁴, R⁵, R⁶ and R⁹ are H,    -   R⁷ and R⁸ are C₁ to C₄ alkyl or phenyl,    -   R^(N1), R^(N2), R^(N3) and R^(N4) are individually unsubstituted        or amino-, hydroxyl- or fluoro substituted C₁ to C₄ alkyl, or        R^(N1) together with R^(N2), and R^(N3) together with R^(N4)        together are —(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅—, —(CH₂)₂O(CH₂)₂— or        —(CH₂)₂NH(CH₂)₂— and    -   R¹⁰ is selected from        -   unsubstituted or amino-, hydroxyl-, carboxyl- and/or fluoro            substituted C₂ to C₁₂ alkyl or C₃ to C₇ cycloalkyl; or        -   R¹⁰ is -L^(A1) _(n)-L^(J1) _(n)′-L^(A2) _(m)-L^(J2)            _(m)′-L^(A3) _(p)-L^(J3) _(p)′-L^(A4) _(q)-L^(J4)            _(q)′-M_(s), wherein L^(A1 . . . 4), L^(J1 . . . 4), n, n′ .            . . q′, s and M have the definitions recited above.

The invention further encompasses the following compounds:

-   -   a.        4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoic        acid (2a);    -   b.        4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-3-methylbutanoic        acid (3a);    -   c.        N³,N³,N⁷,N⁷,5,5-Hexamethyl-10-propylidene-5,10-dihydrodibenzo[b,e]siline-3,7-diamine        (4a);    -   d.        N³,N³,N⁷,N⁷,5,5-Hexamethyl-10-methylene-5,10-dihydrodibenzo[b,e]siline-3,7-diamine        (5a);    -   e.        3-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)propanoic        acid (22);    -   f.        4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-2,2-dimethylbutanoic        acid (28);    -   g.        4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanoic        acid (6a);    -   h.        4-(3,7-Bis(dimethylamino)-2,8-difluoro-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoic        acid (36)    -   i.        3-(3,7-Di(azetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)propanoic        acid (42);    -   j.        4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide        (44);    -   k.        2,5-Dioxopyrrolidin-1-yl-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoate        (45);    -   l.        N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyI)-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide        (47);    -   m. N-(4-((4R,7R,10S,13S,19S,E)-7-((1        H-Indol-2-yl)methyl)-4-(4-hydroxyphenyl)-8,13,15,19-tetramethyl-2,6,9,12-tetraoxo-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl)butyl)-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide        (49);    -   n.        4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(4-(4-(6-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)butyl)butanamide        (51);    -   o.        (2αR,4S,4αS,6R,9S,11S,12S,12αR,12βS)-12β-Acetoxy-9-((3-(4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-4α,8,13,13-tetramethyl-5-oxo-2α,3,4,4α,5,6,9,10,11,12,12α,12β-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-β]oxet-12-yl        benzoate (53);    -   p.        8-(4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)octanoic        acid (55);    -   q.        (2αR,4S,4αS,6R,9S,11S,12S,12αR,12βS)-12β-Acetoxy-9-((3-(8-(4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)octanamido)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-4α,8,13,13-tetramethyl-5-oxo-2α,3,4,4α,5,6,9,10,11,12,12α,12β-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-β]oxet-12-yl        benzoate (56);    -   r.        4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)butanamide        (58);    -   s.        4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide        (59);    -   t.        4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)-N-(21-chloro-8-oxo-3,6,12,15-tetraoxa-9-azahenicosyl)butanamide        (64);    -   u.        1-(4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3,6,9,12-tetraoxapentadecan-15-amide        (65);    -   v.        N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamide        (66);    -   w.        3-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)amino)-2-(4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-3-oxopropane-1-sulfonic        acid (69);    -   x.        N-(4-((4R,7R,10S,13S,19S,E)-7-((1H-Indol-2-yl)methyl)-4-(4-hydroxyphenyl)-8,13,15,19-tetramethyl-2,6,9,12-tetraoxo-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl)butyl)-4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamide        (70);    -   y.        N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-(3,7-bis(dimethylamino)-2,8-difluoro-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide        (71);    -   z.        N-(4-((4R,7R,10S,13S,19S,E)-7-((1H-Indo)-2-yl)methyl)-4-(4-hydroxyphenyl)-8,13,15,19-tetramethyl-2,6,9,12-tetraoxo-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl)butyl)-4-(3,7-bis(dimethylamino)-2,8-difluoro-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide        (72).

Another aspect of the invention relates to the use of a compoundaccording to any one of the preceding aspects and embodiments of theinvention in a method of staining a biological sample.

Further, a method to stain a sample is encompassed by the invention,wherein the method comprising the steps of:

-   -   a. contacting the sample with a compound according to any one of        the preceding aspects,    -   b. illuminating the sample with light of a wavelength ranging        from 280 to 450 nm, particularly in the range of 280-405 nm.    -   c. recording the presence and location of said compound in said        sample by illuminating the sample with an appropriate excitation        wavelength λ, particularly a λ close to the maximum of the        excitation spectrum of 646 nm;    -   d. and recording light emitted from said sample at an        appropriate emission wavelength λ, particularly a λ close to the        maximum of the emission spectrum of 668 nm.

In order to avoid activation of the fluorophore prior to themeasurement, illumination of the dye or the sample containing the dyecompound of the invention with light of the photoactivating wavelengthmust be avoided.

Wherever alternatives for single separable features such as, forexample, R¹, R², R^(N1) or R¹⁰ are laid out herein as “embodiments”, itis to be understood that such alternatives may be combined freely toform discrete embodiments of the invention disclosed herein.

The invention is further illustrated by the following examples andfigures, from which further embodiments and advantages can be drawn.These examples are meant to illustrate the invention but not to limitits scope.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows (A) structures of PA-SiR 1a, the fluorescent cation 1b andthe non-fluorescent compound 1c. (B) Top UV-Vis spectra of PA-SiR 2a (20μm in PBS) before and after activation by UV irradiation (2 min). Thespectra of the activated solution were measured every 1 min. BottomChange of absorbance after activation at four different wavelengths overtime. (C) Excitation and emission spectrum of the cation 2b.

FIG. 2 shows (A) structures of the measured PA-SiR's. (B) Decay ofabsorbance at 646 nm normalized to the initial value reached afterphotoactivation of each compound. Compounds were dissolved in PBS/ACN(10 μM, 7:3, F) except 2a in PBS pH=7.3, 11.1 or 3.1, where the pH wasadjusted by addition of HCl and NaOH. (C) Different PA-SiR probestogether with the measurement on their respective protein-tag (10 μMPBS, M).

FIG. 3 U2OS cells expressing HaloTag-H2B fusion stained with PA-SiR-Halofor 1 h (1 μM) and washed once for 3 min. Images were taken before andafter irradiation with the 355 nm laser on a confocal microscope. Scalebar 30 μm.

EXAMPLES Example 1 General Description of All Species Involved in thisStudy

Photoactivatable silicon rhodamines were synthesised in three steps fromcommercially available starting materials in analogy to a previouslypublished synthesis (Grimm et al., ACS Cent. Sci. 2017, 3, 975-985).

Upon UV irradiation of an aqueous solution of PA-SiR 2a, the fluorescentand blue-coloured cation 2b is forming (FIG. 1A), as confirmed by LC-MS.Compound 2b has an excitation maximum at 646 nm and an emission maximumat 668 nm. The extinction coefficient is 25,000 M⁻¹cm⁻¹ (in PBS at pH7.3) and the quantum yield in aqueous solution was measured to be 0.37,whereas 2a shows only absorbance in the UV-range (FIGS. 1B and C andTable 1). However, the blue solution of 2b undergoes discoloration overtime (FIG. 1C). It was shown by ¹H NMR that the discoloration observedby UV-Vis spectroscopy, which takes place on a time scale of roughly 10min, could be assigned to the formation of a new species. The newspecies corresponds to the hydroxylated form 2c (LC-MS) as thefluorescent cation 2b is attacked by nucleophiles at the electrophiliccentral carbon. Thus, 2b is in equilibrium with the hydroxylated form 2cin aqeous solution, which leads to the discoloration of the bluesolution within 10 min. The thermodynamics and kinetics of thesereactions are discussed in more detail below.

Example 2 Fine-Tuning of the Equilibria Between Fluorescent Cation andthe Non-Fluorescent Compounds

In order to study the kinetic properties of the second equilibrium (FIG.1A, k₂ and k⁻²), UV-Vis spectroscopy was used. In order to achieve thehighest possible time resolution, the photoactivation was carried out inthe spectrometer. These measurements revealed a dependence of thephotoactivation and the second equilibrium between 1b and 1c (bothkinetics and thermodynamics) on pH and solvent polarity (FIG. 2B).Effects such as solubility, pH, nucleophilicity and electrophilicity ofthe involved species play an important role.

Moreover, similar UV-Vis analysis of several analogues illustrated theinfluence on this equilibrium of different substituents at variouspositions (FIG. 2 ). It was shown that the ratio of the fluorescentcation 1b to hydroxylated compound 1c could be tuned in both directionsin comparison to the standard compound 2a by structural changes at theperiphery.

Since both the kinetic properties and the equilibrium of the second stepcould be tuned by chemical modifications and changes in solvent, theinventors hypothesised that also the microenvironment of proteinsurfaces can influence the stability of the fluorescent cation 1 b.Therefore, SNAP-tag and HaloTag substrates were synthesised and reactedwith their respective protein tags. It could be shown that the samefluorophore showed different behaviour on the two protein-tags (FIG.2C). A stable signal was achieved on the surface of HaloTag. Incontrast, with SNAP-tag a fast decay was seen. The difference in decayrates permits to distinguish different molecular species such as SNAPand HaloTag labelled with the same PA-SiR. The decay rate also reportson the environment of the activated fluorophore.

Motivated by the versatility of the compounds the inventors synthesisedcorresponding probes from PA-SIR and various ligands (PA-SiR-Halo,PA-SiR-SNAP, PA-SiR-Actin, PA-SiR-DNA, PA-SiR-Tubulin) and tested themfor applications in live-cell imaging. The olefinic structure is notcharged and therefore ideal to pass the plasma membrane. The compoundsshowed good cell-permeability and could be activated efficiently in livecells in both widefield (DAPI channel) and confocal microscopy (355 nmlaser). PA-SiR-SNAP and PA-SiR-Halo probes were successfully localisedin Hela cells expressing either SNAP-tag or HaloTag in the nucleus.PA-SiR-Halo was also successfully localised to H2B (FIG. 3 ).PA-SiR-Actin, PA-SiR-DNA and PA-SiR-Tubulin were localised to theirtargets.

Example 3 General Procedure A for the Silane Introduction

3-Bromo-N,N-dimethylaniline (13) (3.20 g, 16.00 mmol, 2.0 eq.) wasdissolved in dry Et₂O (45 mL) and cooled down to −78° C. sec-BuLi (14.0mL, 18.40 mmol, 2.3 eq., 1.3 M in cyclohexane) was added dropwise over15 min and the mixture was stirred for 30 min at −78° C.Dichlorodimethylsilane (14) (1.0 mL, 8.00 mmol, 1.0 eq.) was addeddropwise over 10 min at −78° C. The mixture was stirred for 10 min at−78° C. and then warmed up to room temperature and stirred for 1 h. Themixture was quenched with aqueous saturated NaHCO₃ solution. The aqueouslayer was extracted with Et₂O (3×150 mL) and the combined organic layerswere dried over MgSO₄, filtered and evaporated to afford the crudeproduct.

Example 4 General Procedure B for the Bromination

A solution of 15 (1.85 g, 6.18 mmol, 1.0 eq.) and ammonium acetate (95mg, 1.24 mmol, 0.2 eq.) in ACN (30 mL) was cooled down to 0° C. NBS (2.3g, 12.98 mmol, 2.1 eq.) was added portion wise over 10 min. The mixturewas stirred at 0° C. for 30 min and then warmed up to room temperatureand stirred for 2 h. A mixture of aqueous saturated NaHCO₃ solution andwater 1:1 was added. The aqueous layer was extracted with CH₂Cl₂ (3×100mL) and the combined organic layers were dried over MgSO₄, filtered andevaporated to afford the crude product.

Example 5 General Procedure C for the Ring Closure

A solution of 16 (365 mg, 0.8 mmol, 1.0 eq.) in dry THF (8 mL) wascooled down to −78° C. sec-BuLi (1.4 mL, 1.76 mmol, 2.2 eq., 1.3 M incyclohexane) was added dropwise over 5 min and the mixture was stirredfor 30 min at −78° C. A solution of glutaric anhydride (17) (100 mg,0.88 mmol, 1.1 eq.) in dry THF (1.0 mL) was added to the mixture. Themixture was stirred at −78° C. for 15 min and then warmed up to roomtemperature and stirred for 30 min. Acetic acid (2 mL) was added to themixture. The blue mixture was adsorbed on SiO₂ (2 g).

Example 6 Materials and Methods

All chemical reagents and anhydrous solvents for synthesis werepurchased from commercial suppliers (Acros, Apollo, Armar, Bachchem,Biomatrik, Fluka, Fluorochem, LC Laboratories, Merck, Reseachem, Roth,Sigma-Aldrich and TCI) and used without further purification. BG-NH₂ 48and Halo-NHBoc 43 were available in the Johnsson group.Jasplakinolide-NHBoc 48 was obtained from a custom synthesis.Composition of mixed solvents is given by volume ratio (v/v). Reactionsin the absence of air and moisture were performed in oven-driedglassware under Ar or N2 atmosphere. Flash column chromatography wasperformed using a CombiFlash Rf system (Teledyne ISCO) using SiO₂RediSep® Rf columns at 25° C. The used solvent compositions are reportedindividually in parentheses. Analytical thin layer chromatography wasperformed on glass plates coated with silica gel 60 F254 (Merck).Visualisation was achieved using UV light (254 nm). Evaporation in vacuowas performed at 25-60° C. and 900-10 mbar. ¹H, ¹³C, and ¹⁹F NMR spectrawere recorded on AV 400 and AV 600 Bruker spectrometers at 400 MHz or600 MHz (¹H), 101 MHz or 151 MHz (¹³C), 377 MHz or 566 MHz (¹⁹F)respectively. All spectra were recorded at 298 K. Chemical shifts δ arereported in ppm downfield from tetramethylsilane using the residualdeuterated solvent signals as an internal reference (CDCl₃: δ_(H)=7.26ppm, δ_(C)=77.16 ppm; CD₃OD: δ_(H)=3.31 ppm, δ_(C)=49.00 ppm; DMSO-d₆:δ_(H)=2.50 ppm, δ_(C)=39.52 ppm; ACN-d₃: δ_(H)=1.94 ppm, δ_(C)=118.26ppm). For ¹H, ¹³C and ¹⁹F NMR, coupling constants J are given in Hz andthe resonance multiplicity is described as s (singlet), d (doublet), t(triplet), q (quartet), quint (quintet), sext (sextet), sept (septet), m(multiplet) and br. (broad). High-resolution mass spectrometry (HRMS)was performed by the MS-service of the EPF Lausanne (SSMI) on a WatersXevo® G2-S Q-Tof spectrometer with electron spray ionisation (ESI).Liquid chromatography coupled to mass spectrometry (LC-MS) was performedon a Shimadzu MS2020 connected to a Nexera UHPLC system equipped with aWaters ACQUITY UPLC BEH C18 (1.7 μm, 2.1×50 mm) column. Buffer A: 0.05%HCOOH in H₂O Buffer B: 0.05% HCOOH in ACN. Analytical gradient was from10% to 90% B within 6 min with 0.5 mL/min flow unless otherwise stated.Preparative reverse phase high-performance liquid chromatography(RP-HPLC) was carried out on a Dionex system equipped with an UltiMate3000 diode array detector for product visualisation on a Waters SymmetryC18 column (5 μm, 3.9×150 mm) or on a Waters SunFire™ Prep C18 OBD™ (5μm, 10×150 mm) column. Buffer A: 0.1% TFA in H₂O Buffer B: ACN. Typicalgradient was from 10% to 90% B within 32 min with 3 or 4 mL/min flow.

Example 7 In Vitro Characterization and Microscopy

General Considerations

Three different UV light sources were used in the following experiments:a transilluminator (Biometra TI 1, 312 nm, high), a photography flash(Agfatronic 261B, plastic cover removed), a monochromator (Polychrome V,FEI, at 330 nm for 12 s) abbreviated as T, F and M. PBS (6.7 mm, Lonza)was used in all experiments.

¹H NMR Analysis

PA-SiR 2a (1 mg, 2.5 pmol) was dissolved in PBS/D₂O (1 mL, 90:10) andNaOH (1 μL, 5 M) was added to achieve better solubility as PA-SiR 2a wasisolated as its TFA salt (pH=7-8, pH paper). ¹H NMR spectra weremeasured on a Bruker AV 600 spectrometer at 600 MHz and 298 K. Chemicalshifts δ are reported in ppm downfield from tetramethylsilane using theDMSO signal (δ_(H)=2.50 ppm) instead of the residual deuterated solventsignal as an internal reference. Spectra were measured with either NS=128 using a water suppression pre-saturation sequence. UV irradiationwas performed outside of the spectrometer for the indicated times (T).After each irradiation step the NMR sample was transferred to the NMRspectrometer.

LC-MS Analysis

PA-SiR 2a was dissolved in water (50 pM). UV irradiation was performedin a quartz cuvette (Hellma Analytics) for the indicated times (T).Aliquots were taken to measure LC-MS at defined time points using ananalytical gradient from 10% to 100% B within 6 min with 0.5 mL/minflow.

Fluorescence Measurements and Determination of Quantum Yield

Fluorescence spectra were measured on an Infinite M1000 (Tecan) platereader. Quantum yields were determined using a Quantaurus QY(Hamamatsu).

UV-Vis Analysis

PA-SiRs were prepared as stock solutions in dry DMSO and diluted in PBSor PBS/ACN such that the final concentration of DMSO did not exceed 5%v/v. PBS solutions of different pH were adjusted by addition of HCI orNaOH solution using a pH meter.

Full Absorbance Spectra Measurements of PA-SiR 2a

Solutions were prepared in PBS (10 μm or 20 μM) at the indicated pH. UVirradiation was performed outside of the spectrometer for the indicatedperiod (T). Absorbance spectra were recorded using a SHIMADZU UVspectrophotometer (UV-1800) and 1 cm fluorescence quartz cuvettes(Hellma Analytics). Spectra were recorded every minute.

pH Dependence of PA-SiR 2a

Solutions of 2a in PBS (10 μM) at the desired pH were prepared in 1 cmfluorescence quartz cuvette (Hellma Analytics). UV irradiation wasperformed directly inside the spectrophotometer during the runningexperiment at a fixed distance (F, single flash). Kinetic absorbancemeasurements were recorded on a Perkin-Elmer Lambda 950spectrophotometer at 646 nm.

UV-Vis Analysis of Various PA-SiRs

Solutions were prepared in PBS/ACN (10 μM, 7:3) in 1 cm fluorescencequartz cuvettes (Hellma Analytics). UV irradiation was performeddirectly inside the spectrophotometer during the running experiment at afixed distance (F, single flash). Kinetic absorbance measurements wererecorded on a Perkin-Elmer Lambda 950 spectrophotometer at 646 nm.

UV-Vis Analysis of PA-SiR-Halo and PA-SiR-SNAP

SNAP-tag protein and HaloTag protein were available in the Johnsson labas 5.3 and 3.6 mm solutions in HEPES-Glycerol (1:1). PA-SiR-Halo andPA-SiR-SNAP were dissolved in PBS (10 μM) and protein (20 μM, 2.0equiv.) was added. The mixture was incubated for 1 h (HaloTag) or 2 h(SNAP-tag) before UV-Vis measurement. UV irradiation was performeddirectly inside the spectrophotometer during the running experiment at afixed distance (M, 12 s). Kinetic absorbance measurements were recordedon a Jasco V770 spectrophotometer equipped with a Peltier coolingelement (PAC743R) at 646 nm.

Plasmids

Mammalian expression vectors with a SNAP-tag-NLS orSNAP-tag-Halo-tag-NLS fusion construct were available in the Johnssonlab. H2B-Halo was constructed from a pEBTet plasmid and the commerciallyavailable pCLIPf-H2B plasmid (NEB).

Cell Culture and Transfection

HeLa or U2OS cells were cultured in high-glucose DMEM media withGlutaMAX-1 (Life Technologies) supplemented with 10% FBS (LifeTechnologies) in a humidified 5% CO₂ incubator at 37° C. Cells weresplit every 3-4 days or at confluency.

Cells were seeded on glass bottom 35 mm dishes (Mattek) one day beforeimaging. Transient transfection of cells was performed usingLipofectamine™ 2000 reagent (Life Technologies) according to themanufacturer's recommendations: DNA (2.5 μg) was mixed with OptiMEM I(100 μL, Life Technologies) and Lipofectamine™ 2000 (6 μL) was mixedwith OptiMEM I (100 μL). The solutions were incubated for 5 min at roomtemperature. They were mixed and incubated for 20 min at roomtemperature. Prepared DNA-Lipofectamine complex was added to a glassbottom 35 mm dish with cells at 50-70% confluency. After 6 h incubationin a humidified 5% CO₂ incubator at 37° C. the medium was changed to afresh high-glucose DMEM medium with GlutaMAX-1 supplemented with 10%FBS. The cells were incubated for 1-2 days before imaging.

Staining

Cells were stained with 1-3 μM PA-SiR (1-2 h, 37° C.), washed withphenol-red free DMEM medium (Life Technologies) or PBS (once for 3 min,37° C.) and imaged in the same medium.

Widefield Microscopy

Imaging was performed using a Leica DMI6000B microscope equipped with aHamamatsu-C9100 EM-CCD camera and a HCX PL APO 100.0×1.47 Oil objectiveand a standard Cy5 filter set was used. Activation of the fluorophoreswas achieved by irradiation with the DAPI-channel at 100% for 10-20 s.The same settings (exposure time=150 ms, gain=3, EM-gain=800,transmission=12.5%) were used for the images taken before and after UVirradiation in the SiR-channel.

Confocal Microscopy

Confocal imaging was performed on a Leica DMi8 microscope equipped witha Leica TCS SP8 X scanhead, a white light laser, a 355 nm laser(Coherent) and HC PL APO 63×1.47 Oil objective. The 355 nm laser wasused to perform the photoactivation.

All images were processed with Fiji.

Example 9 Synthesis 3,3′-(Dimethylsilanediyl)bis(N,N-dimethylaniline)(15)

Following general procedure, A, flash column chromatography (SiO₂,hexane/EtOAc 100:0→70:30) gave 15 (1.598 g, 67%) as a colourless oil.

¹H NMR (400 MHz, CDCl₃) δ=0.56 (s, 6H), 2.94 (s, 12H), 6.78 (ddd, J=8.3,2.8, 1.0 Hz, 2H), 6.92-6.98 (m, 4H), 7.22-7.31 ppm (m, 2H); ¹³C NMR (101MHz, CDCl₃) δ=−2.01, 40.82, 113.68, 117.28, 118.46, 121.49, 122.86,128.60, 139.07, 150.05 ppm; HRMS (ESI): m/z calcd for C₁₈H₂₇N₂Si⁺ [M+H]⁺299.1938, found 299.1940; LCMS (LC, 10% to 90%): t_(R)=3.31 min.

3,3′-(Dimethylsilanediyl)bis(4-bromo-N,N-dimethylaniline) (16)

Following general procedure B, flash column chromatography (SiO₂,hexane/CH₂Cl₂ 100:0→0:100) gave 16 (2.270 g, 80%) as a beige solid.

¹H NMR (400 MHz, CDCl₃) δ =0.75 (s, 6H), 2.88 (s, 12H), 6.60 (dd, J=8.7,3.2 Hz, 2H), 6.84 (d, J=3.2 Hz, 2H), 7.35 ppm (d, J=8.7 Hz, 2H); ¹³C NMR(101 MHz, CDC₃) δ=−0.79, 40.71, 115.40, 116.94, 121.92, 133.10, 138.87,149.03 ppm; HRMS (ESI): m/z calcd for C₁₈H₂₅Br₂N₂Si⁺ [M+H]⁺ 455.0148,found 455.0145; LCMS (LC, 10% to 100%): t_(R)=5.18 min.

4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoicacid(2a)

Following general procedure C, flash column chromatography (SiO₂,CH₂Cl₂/MeOH 100:0→90:10) and RP-HPLC (3 mL/min, 10% to 90% B in 32 min)gave 2a (23 mg, 7%) as a green solid.

¹H NMR (400 MHz, CD₃OD) δ=0.50 (s, 6H), 2.44 (t, J=7.2 Hz, 2H), 2.68 (d,J=7.4 Hz, 2H), 3.21 (s, 6H), 3.24 (s, 6H), 5.98 (t, J=7.3 Hz, 1H), 7.40(ddd, J=39.8, 8.5, 2.7 Hz, 2H), 7.51-7.69 ppm (m, 4H); ¹³C NMR (101 MHz,CD₃OD) δ=−1.11, 26.63, 34.76, 45.68, 46.39, 120.02, 121.75, 123.67,123.77, 128.57, 131.34, 134.12, 138.48, 140.26, 140.97, 143.25, 143.43,144.46, 150.16, 176.34 ppm; HRMS (ESI): m/z calcd forC₂₃H₂₉N₂O₂Si⁻[M−H]³¹ 393.2004, found 393.1992; LCMS (LC, 10% to 100%):t_(R)=2.58 min.

4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-3-methylbutanoicacid (3a)

Following general procedure C, flash column chromatography (SiO₂,CH₂Cl₂/MeOH 100:0→90:10) and RP-HPLC (3 mL/min, 10% to 65% B in 32 min)gave 3a (0.9 mg, 0.6%) as a light blue solid.

¹H NMR (400 MHz, DMSO-d₆) δ=0.37 (s, 6H), 1.04 (d, J=7.0 Hz, 3H), 2.26(br. s, 3H, overlaps with the residual solvent signal), 2.52 (s, 1H,overlaps with the residual solvent signal), 2.92-2.97 (m, 12H), 5.51 (d,J=10.3 Hz, 1H), 6.77-6.95 (m, 2H), 7.03 (s, 2H), 7.21-7.38 ppm (m, 2H);HRMS (ESI): m/z calcd for C₂₄H₃₁N₂O₂Si⁻ [M−H]⁻ 407.2160, found 407.2155;LCMS (LC, 10% to 100%): t_(R)=2.64 min.

N³,N³,N⁷,N⁷,5,5-Hexamethyl-10-propylidene-5,10-dihydrodibenzo[b,e]siline-3,7-diamine(4a)

Following general procedure C, flash column chromatography (SiO₂,hexane/EtOAc 90:10→70:30) and RP-HPLC (3 mL/min, 10% to 100% B in 32min) gave 4a (61 mg, 35%) as a white solid.

¹H NMR (400 MHz, CDCl₃) δ=0.42 (s, 6H), 1.02 (t, J=7.4 Hz, 3H), 2.40 (p,J=7.4 Hz, 2H), 2.94 (s, 6H), 2.97 (s, 6H), 5.73 (t, J=7.3 Hz, 1H), 6.74(td, J=8.4, 2.8 Hz, 2H), 6.91 (d, J=2.9 Hz, 1H), 6.97 (d, J=2.8 Hz, 1H),7.29 (d, J=8.5 Hz, 1H), 7.39 ppm (d, J=8.5 Hz, 1H).

N³,N³,N⁷,N⁷,5,5-Hexamethyl-10-methylene-5,10-dihydrodibenzo[b,e]siline-3,7-diamine(5a)

Following general procedure C, washing of the crystals with MeOH gave 5a(195 mg, 76%) as a colourless solid.

¹H NMR (400 MHz, CDCl₃) δ=0.44 (s, 6H), 2.99 (s, 12H), 5.41 (s, 2H),6.79 (dd, J=8.7, 2.8 Hz, 2H), 6.93 (d, J=2.8 Hz, 2H), 7.59 ppm (d, J=8.7Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ=−2.14, 40.85, 111.33, 114.17,115.88, 126.93, 134.83, 135.20, 147.69, 149.17 ppm; HRMS (ESI): m/zcalcd for C₂₀H₂₇N₂Si⁺ [M+H]⁺ 323.1938, found 323.1936; LCMS (LC, 10% to100%): t_(R)=3.79 min.

3-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)propanoicacid (22)

Following general procedure C, flash column chromatography (SiO₂,DCM/MeOH 100:0→90:10) gave 22 (46 mg, 24%) as a green solid.

¹H NMR (400 MHz, CD₃CN) δ=0.36-0.50 (br. s, 6H), 3.09 (s, 6H), 3.12 (s,6H), 3.34 (d, J=7.5 Hz, 2H), 6.06 (t, J=7.5 Hz, 1H), 7.25 (dd, J=8.5,2.7 Hz, 1H), 7.41 (dd, J=8.5, 2.7 Hz, 1H), 7.44 (d, J=8.5 Hz, 1H), 7.50(d, J=2.7 Hz, 1H), 7.55-7.62 ppm (m, 2H); ¹³C NMR (101 MHz, CD₃CN)δ=35.56, 44.04, 45.48, 121.04, 122.09, 123.23, 125.15, 127.88, 130.45,137.61, 138.75, 139.07, 142.38, 144.34, 146.14, 147.53, 172.88 ppm (twosignals are hidden by the residual solvent signal).

3,7-Bis(dimethylamino)-4′,4′,5,5-tetramethyl-3′,4′-dihydro-5H,5′H-spiro[dibenzo[b,e]siline-10,2′-furan]-5′-one(24)

Following general procedure C, flash column chromatography (SiO₂,hexane/EtOAc 70:30→0:100) and RP-HPLC (3 mL/min, 10% to 100% B in 32min) gave 24 (8.4 mg, 4%) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ=0.45 (s, 3H), 0.59 (s, 3H), 1.10 (s, 6H),2.28 (s, 2H), 2.96 (s, 12H), 6.87-6.97 (m, 2H), 7.09-7.20 (m, 2H), 7.33ppm (d, J=8.7 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ=−2.83, 0.35, 26.86,NMe₂ hidden by the residual solvent signal, 40.93, 56.27, 83.83, 114.08,116.97, 122.44, 133.63, 141.72, 147.69, 182.28 ppm; HRMS (ESI): m/zcalcd for C₂₄H₃₃N₂O₂Si⁺ [M+H]⁺ 409.2306, found 409.2304; LCMS (LC, 10%to 100%): t_(R)=3.95 min.

3,7-Bis(dimethylamino)-10-isopropyl-5,5-dimethyl-5,10-dihydrodibenzo[b,e]silin-10-ol(26)

Following general procedure C, flash column chromatography (SiO₂,hexane/EtOAc 90:10→50:50) gave 26 (68 mg, 38%) as a white solid.

¹H NMR (400 MHz, CDCl₃) δ=0.45 (s, 3H), 0.48 (s, 3H), 0.70 (d, J=6.8 Hz,6H), 2.97 (s, 12H), 6.82 (dd, J=8.8, 2.9 Hz, 2H), 6.93 (d, J=2.9 Hz,2H), 7.75 ppm (d, J=8.8 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ=−0.62, 0.57,18.19, 40.85, 43.42, 79.44, 113.52, 117.00, 127.22, 134.53, 141.39,148.52 ppm.

4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-2,2-dimethylbutanoicacid (28)

Following general procedure C, flash column chromatography (SiO₂,hexane/EtOAc 100:0→0:100) gave 28 (12 mg, 6%) as a green solid.

¹H NMR (400 MHz, CDCl₃) δ=0.41 (s, 6H), 1.16 (s, 6H), 2.71 (d, J=7.1 Hz,2H), 2.94 (s, 6H), 2.98 (s, 6H), 5.73 (t, J=7.1 Hz, 1H), 6.74 (ddd,J=8.5, 6.7, 2.8 Hz, 2H), 6.91 (d, J=2.8 Hz, 1H), 6.96 (d, J=2.8 Hz, 1H),7.26 (d, J=8.5 Hz, 1H), 7.38 ppm (d, J=8.5 Hz, 1H).

3,3′-(Diisopropylsilanediyl)bis(N,N-dimethylaniline) (30)

Following general procedure A, flash column chromatography (SiO₂,hexane/EtOAc 100:0→80:20) gave 30 (0.440 g, 31%) as a colourless oil.

¹H NMR (400 MHz, CDCl₃) δ=0.99 (d, J=7.4 Hz, 12H), 1.56 (hept, J=7.3 Hz,2H), 2.93 (s, 12H), 6.81 (ddd, J=8.3, 2.7, 1.0 Hz, 2H), 6.91-7.01 (m,4H), 7.21-7.31 ppm (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ=10.06, 17.82,40.96, 113.65, 120.91, 125.16, 128.13, 133.91, 149.74 ppm; HRMS (ESI):m/z calcd for C₂₂H₃₅N₂Si⁺ [M+H]⁺ 355.2564, found 355.2567; LCMS (LC, 10%to 100%): t_(R)=4.87 min.

3,3′-(Diisopropylsilanediyl)bis(4-bromo-N,N-dimethylaniline) (31)

Following general procedure B, flash column chromatography (SiO₂,hexane/CH₂Cl₂ 100:0→0:100) gave 31 (1.087 g, 73%) as a white solid.

¹H NMR (400 MHz, CDCl₃) δ=1.17 (d, J=7.5 Hz, 12H), 2.00 (p, J=7.4 Hz,2H), 2.92 (s, 12H), 6.60 (dd, J=8.8, 3.3 Hz, 2H), 6.97 (d, J=3.2 Hz,2H), 7.33 ppm (d, J=8.7 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ=12.68,19.12, 40.80, 115.10, 117.63, 122.75, 133.40, 136.75, 148.57 ppm; HRMS(ESI): m/z calcd for C₂₂H₃₃Br₂N₂Si⁺ [M+H]⁺ 511.0774, found 511.0779;LCMS (LC, 10% to 100%): t_(R)=5.73 min.

4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanoicacid (6a)

Following general procedure C, flash column chromatography (SiO₂,hexane/EtOAc 100:0→50:50) and RP-HPLC (3 mL/min, 10% to 90% B in 32 min)gave 6a (6.7 mg, 7%) as a light blue solid.

¹H NMR (400 MHz, CD₃OD) δ=1.09 (dd J=7.7 Hz, 12H), 1.50-1.65 (m, 2H),2.41 (t, J=7.4 Hz, 2H), 2.65 (q, J=7.6 Hz, 2H), 3.20 (s, 6H), 3.24 (s,6H), 5.90 (t, J=7.2 Hz, 1H), 7.37 (dd, J=8.6, 2.7 Hz, 1H), 7.48-7.61 (m,4H), 7.69 ppm (d, J=8.5 Hz, 1H); ¹³C NMR (101 MHz, CD₃OD) δ=12.87,18.43, 26.94, 34.84, 44.48, 45.82, 118.48, 120.96, 122.99, 123.90,128.71, 131.61, 133.71, 134.66, 135.99, 141.75, 142.16, 143.85, 145.71,150.46, 176.48 ppm; HRMS (ESI): m/z calcd for C₂₇H₃₇N₂O₂Si⁻ [M−H]⁻449.2630, found 449.2622; LCMS (LC, 10% to 90%): t_(R)=3.92 min.

5-Bromo-2-fluoro-N,N-dimethylaniline (33)

A solution of 5-bromo-2-fluoroaniline (32) (5.0 g, 26.3 mmol, 1.0 eq.)in MeOH (30 mL) was treated with acetic acid (40 mL) andparaformaldehyde (3.9 g, 131.6 mmol, 5.0 eq.). The mixture was cooleddown to 0° C. and stirred for 15 min. NaBH₃CN (5.0 g, 78.9 mmol, 3.0eq.) was added portion wise to the mixture over 10 min. The mixture waswarmed up to room temperature and was stirred for 16 h. The mixture wasevaporated and then neutralised with aqueous NaOH solution (4 mL, 5 M).The aqueous layer was extracted with CH₂Cl₂ (3×100 mL). The combinedorganic layers were dried over MgSO₄, filtered and evaporated to affordthe crude product. Flash column chromatography (SiO₂, hexane/EtOAc100:0→85:15) gave 33 (3.990 g, 70%) as a yellow oil.

¹H NMR (400 MHz, CDCl₃) δ=2.85 (d, J=1.0 Hz, 6H), 6.86 (dd, J=12.6, 8.4Hz, 1H), 6.90-6.99 ppm (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ=42.68 (d,J=4.5 Hz), 116.87 (d, J=3.3 Hz), 117.58 (d, J=22.8 Hz), 121.18 (d, J=3.9Hz), 123.28 (d, J=7.9 Hz), 142.10 (d, J=9.7 Hz), 154.07 ppm (d, J=245.3Hz); ¹⁹F NMR (376 MHz, CDCl₃) δ=−124.69-−124.58 ppm (m); HRMS (ESI): m/zcalcd for C₈H₁₀BrFN⁺ [M+H]⁺ 217.9975, found 217.9978 ; LCMS (LC, 10% to90%): t_(R)=4.09 min.

5,5′-(Dimethylsilanediyl)bis(2-fluoro-N,N-dimethylaniline) (34)

Following general procedure A, flash column chromatography (SiO₂,hexane/EtOAc 100:0→85:15) gave 34 (2.110 g, 73%) as a yellow oil.

¹H NMR (400 MHz, CDCl₃) δ=0.52 (s, 6H), 2.83 (d, J=0.9 Hz, 12H),6.98-7.06 ppm (m, 6H); ¹³C NMR (101 MHz, CDCl₃) δ=−1.86, 43.01 (d, J=3.9Hz), 115.92 (d, J=19.8 Hz), 123.97 (d, J=3.4 Hz), 127.57 (d, J=7.5 Hz),134.00 (d, J=4.3 Hz), 140.33 (d, J=8.0 Hz), 156.31 ppm (d, J=248.0 Hz);¹⁹F NMR (376 MHz, CDCl₃) δ=−121.34-−121.21 ppm (m); HRMS (ESI): m/zcalcd for C₁₈H₂₅F₂N₂Si⁺ [M+H]⁺ 335.1750, found 335.1754; LCMS (LC, 10%to 90%): t_(R)=4.89 min.

5,5′-(Dimethylsilanediyl)bis(4-bromo-2-fluoro-N,N-dimethylaniline) (35)

Following general procedure B, flash column chromatography (SiO₂,hexane/CH₂Cl₂ 100:0→0:100) gave 35 (2.329 g, 78%) as a beige solid.

¹H NMR (400 MHz, CDCl₃) δ=0.74 (s, 6H), 2.80 (d, J=1.0 Hz, 12H), 6.94(d, J=10.2 Hz, 2H), 7.20 ppm (d, J=12.5 Hz, 2H); ¹³C NMR (101 MHz,CDCl₃) δ=−0.80, 42.70 (d, J=4.0 Hz), 119.91 (d, J=8.6 Hz), 120.90 (d,J=23.2 Hz), 126.70 (d, J=4.0 Hz), 134.08 (d, J=4.0 Hz), 139.37 (d, J=7.5Hz), 155.57 ppm (d, J=252.7 Hz); ¹⁹F NMR (376 MHz, CDCl₃) δ=−118.89 ppm(t, J=11.3 Hz); HRMS (ESI): m/z calcd for C₁₈H₂₃Br₂F₂N₂Si⁺ [M+H]⁺490.9960, found 490.9954; LCMS (LC, 10% to 100%): t_(R)=5.50 min.

4-(3,7-Bis(dimethylamino)-2,8-difluoro-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoicacid (36)

Following general procedure C, flash column chromatography (SiO₂,hexane/EtOAc 80:20→0:100) and RP-HPLC (3 mL/min, 10% to 90% B in 32 min)gave 36 (24 mg, 27%) as a white solid.

¹H NMR (400 MHz, ACN-d₃) δ=0.45 (s, 6H), 2.45 (t, J=7.1 Hz, 2H),2.61-2.68 (m, 2H), 3.08 (d, J=12.9 Hz, 12H), 5.98 (t, J=7.2 Hz, 1H),7.33 (ddd, J=13.5, 12.1, 1.1 Hz, 2H), 7.50-7.63 ppm (m, 2H); ¹³C NMR(101 MHz, CD₃OD) δ=−1.27, 26.48, 34.67, 44.58 (d, J=3.2 Hz), 45.20 (d,J=2.8 Hz), 115.24 (d, J=20.2 Hz), 117.95 (d, J=20.4 Hz), 124.85, 133.20(d, J=4.2 Hz), 133.95, 134.29, 134.82 (d, J=4.3 Hz), 139.92, 140.07,142.25 (d, J=6.8 Hz), 149.66 (d, J=8.1 Hz), 155.32 (d, J=87.3 Hz),157.79 (d, J=87.8 Hz), 176.37 ppm; ¹⁹F NMR (376 MHz, ACN-d₃)δ=−122.40-−122.10 (m), −122.07-−120.43 ppm (m); HRMS (ESI): m/z calcdfor C₂₃H₂₇F₂N₂O₂Si⁻ [M−H]⁻ 429.1815, found 429.1816; LCMS (LC, 10% to90%): t_(R)=4.22 min.

1-(3-Bromophenyl)azetidine (39)

A solution of 1,3-dibromobenzene (38) (2.3 g, 10.0 mmol, 1.5 eq.),Pd₂dba₃ (305 mg, 0.3 mmol, 0.05 eq.), xantphos (387 mg, 0.7 mmol, 0.1eq.) and NaO^(t)Bu (1.9 g, 20.0 mmol, 3.0 eq.) in 1,4-dioxane (50 mL)was degassed with argon for 5 min. Azetidine (37) (452 μL, 6.7 mmol, 1.0eq.) was added and the mixture was again degassed with argon for 10 min.The mixture was stirred at 100° C. for 2 h. The mixture was let cooldown to room temperature and was diluted with EtOAc and aqueoussaturated NaHCO₃ solution. The organic layer was washed with aqueoussaturated NaHCO₃ (2×50 mL), brine, dried over MgSO₄, filtered andevaporated to afford the crude product. Flash column chromatography(SiO₂, hexane/EtOAc 100:0→80:20) gave 39 (1.224 g, 86%) as a yellow oil.

¹H NMR (400 MHz, CDCl₃) δ=2.37 (p, J=7.3 Hz, 2H), 3.87 (t, J=7.3 Hz,4H), 6.34 (ddd, J=8.1, 2.2, 0.9 Hz, 1H), 6.56 (t, J=2.1 Hz, 1H), 6.82(ddd, J=7.9, 1.9, 0.9 Hz, 1H), 7.04 ppm (t, J=8.0 Hz, 1H); ¹³C NMR (101MHz, CDCl₃) δ=16.99, 52.42, 109.93, 114.17, 120.00, 123.14, 130.29,153.32 ppm; HRMS (ESI): m/z calcd for C₉H₁₁BrN⁺ [M+H]⁺ 212.0069, found221.0065; LCMS (LC, 10% to 90%): t_(R)=4.17 min.

Bis(3-(azetidin-1-yl)phenyl)dimethylsilane (40)

Following general procedure A, flash column chromatography (SiO₂,hexane/EtOAc 100:0→60:40) gave 40 (0.473 g, 54%) as a colourless oil.

¹H NMR (400 MHz, CDCl₃) δ=0.51 (s, 6H), 2.34 (p, J=7.2 Hz, 4H), 3.86 (t,J=7.2 Hz, 8H), 6.47 (ddd, J=8.1, 2.4, 0.8 Hz, 2H), 6.61 (d, J=2.1 Hz,2H), 6.90 (dt, J=7.2, 1.1 Hz, 2H), 7.20 ppm (t, J=7.6 Hz, 2H); ¹³C NMR(101 MHz, CDCl₃) δ=−2.09, 17.17, 52.61, 112.27, 116.87, 123.43, 128.35,138.92, 151.64 ppm; HRMS (ESI): m/z calcd for C₂₀H₂₇N₂Si⁺ [M+H]⁺323.1938, found 323.1944; LCMS (LC, 10% to 90%): t_(R)=4.88 min.

Bis(5-(azetidin-1-yl)-2-bromophenyl)dimethylsilane (41)

Following general procedure B, flash column chromatography (SiO₂,hexane/EtOAc 100:0→80:20) gave 41 (0.085 g, 38%) as a beige solid.

¹H NMR (400 MHz, CDCl₃) δ=0.71 (s, 6H), 2.33 (p, J=7.2 Hz, 4H), 3.81 (t,J=7.2 Hz, 8H), 6.31 (dd, J=8.5, 2.9 Hz, 2H), 6.51 (d, J=3.0 Hz, 2H),7.31 ppm (d, J=8.5 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ=−0.89, 17.04,52.59, 114.15, 117.54, 120.40, 132.95, 138.93, 150.62 ppm; HRMS (ESI):m/z calcd for C₂₀H₂₅Br₂N₂Si⁺ [M+H]⁺ 479.0148, found 479.0146; LCMS (LC,10% to 100%): t_(R)=5.40 min.

3-(3,7-Di(azetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)propanoicacid (42)

A solution of 41 (91 mg, 0.19 mmol, 1.0 eq.) in dry THF (3 mL) wascooled down to −78° C. sec-BuLi (0.4 mL, 0.56 mmol, 3.0 eq., 1.3 M incyclohexane) was added dropwise over 5 min and the mixture was stirredfor 30 min at −78° C. A solution of succinic anhydride (21) (21 mg, 0.21mmol, 1.1 eq.) in dry THF (1.0 mL) was added to the mixture. The mixturewas stirred at −78° C. for 15 min and then warmed up to room temperatureand stirred for 30 min. Saturated ammonium chloride solution was addedand extracted with EtOAc (2×25 mL). The combined organic layers weredried over MgSO₄, filtered and evaporated to afford the crude product.Flash column chromatography (SiO₂, hexane/EtOAc 90:10→70:30) and RP-HPLC(3 mL/min, 10% to 90% B in 32 min) gave 42 (0.63 mg, 0.8%) as a lightblue solid.

¹H NMR (400 MHz, CD₃CN) δ=0.33 (m, 6H), 2.34-2.37 (m, 4H, hidden byresidual solvent signal), 3.28 (d, J=6.8 Hz, 2H), 3.89 (td, J=7.2, 3.3Hz, 8H), 5.80 (t, J=7.6 Hz, 1H), 6.49 (ddd, J=11.0, 8.3, 2.6 Hz, 2H),6.68 (d, J=2.6 Hz, 1H), 6.73 (d, J=2.5 Hz, 1H), 7.22 (d, J=8.3 Hz, 1H),7.32 ppm (d, J=8.4 Hz, 1H).

4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide(44, PA-SiR-Halo)

A solution of 2a (1.8 mg, 4.5 μmol, 1.0 eq.) in DMSO (250 μL) wastreated with DIEA (2.2 μL, 13.5 μmol, 3.0 eq.) and TSTU (1.6 mg, 5.4μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature.In a separate vial a solution of Halo-NHBoc 43 (2.0 mg, 6.2 μmol, 1.4eq.) in CH₂Cl₂/TFA (8:2, 80 μL) was shaken for 5 min. The solution wasevaporated and dried on the high vacuum for 1 h. The residue was takenup in DMSO (50 μL) and added to the other mixture. The mixture wasshaken for 10 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min,10% to 90% B in 32 min) gave 44 (1.3 mg, 48%) as a light blue solid.

¹H NMR (400 MHz, CD₃OD) δ=0.45 (s, 6H), 1.30-1.47 (m, 4H), 1.56 (p,J=6.8 Hz, 2H), 1.73 (p, J=6.9 Hz, 2H), 2.34 (t, J=7.3 Hz, 2H), 2.69 (q,J=7.3 Hz, 2H), 3.10 (s, 6H), 3.14 (s, 6H), 3.16-3.26 (m, 2H), 3.33 (d,J=5.7 Hz, 3H, overlaps with the residual solvent signal), 3.40-3.52 (m,8H), 5.85 (t, J=7.2 Hz, 1H), 7.09 (d, J=8.6 Hz, 1H), 7.22 (d, J=8.4 Hz,1H), 7.28 (s, 1H), 7.35 (s, 1H), 7.41 (d, J=8.5 Hz, 1H), 7.53 (d, J=8.5Hz, 1H), 8.02-8.12 ppm (m, 1H); HRMS (ESI): m/z calcd forC₆₃H₅₁ClN₃O₃Si⁺ [M+H]⁺ 600.3383, found 600.3386; LCMS (LC, 10% to 100%):t_(R)=3.61 min.

2,5-Dioxopyrrolidin-1-yl-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoate(45)

A solution of 2a (5.1 mg, 13.0 μmol, 1.0 eq.) in DMSO (300 μl) wastreated with DIEA (6.0 μL, 39 μmol, 3.0 eq.) and TSTU (4.7 mg, 15.6μmol, 1.2 eq.). The mixture was shaken for 30 min at room temperatureand then acidified with TFA (2 μL). RP-HPLC (3 mL/min, 0% to 90% B in 32min) gave 45 (2.1 mg, 33%) as a light green solid.

¹H NMR (400 MHz, CD₃OD) δ=0.51 (br. s, 6H), 2.77-2.89 (m, 8H), 3.21 (s,6H), 3.26 (s, 6H), 5.98-6.09 (m, 1H), 7.31 (dd, J=8.4, 2.8 Hz, 1H), 7.46(dd, J=8.4, 2.7 Hz, 1H), 7.51 (d, J=2.7 Hz, 1H), 7.55 (d, J=8.5 Hz, 1H),7.59 (d, J=2.7 Hz, 1H), 7.68 ppm (d, J=8.5 Hz, 1H); ¹³C NMR (101 MHz,CD₃OD) δ=−1.78, 24.76, 25.08, 30.31, 42.65, 44.22, 116.61, 119.21,120.15, 121.18, 127.08, 129.51, 133.26, 136.90, 138.08, 138.48, 139.02,140.84, 141.30, 142.95, 168.29, 170.40 ppm; HRMS (ESI): m/z calcd forC₂₇H₃₄N₃O₄Si⁺ [M+H]⁺ 492.2312, found 492.2311; LCMS (LC, 10% to 90%):t_(R)=3.50 min.

N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide(47, PA-SiR-SNAP)

A solution of 45 (1.2 mg, 3.0 μmol, 1.0 eq.) in DMSO (100 μl) wastreated with DIEA (1.5 μL, 9 μmol, 3.0 eq.) and BG-NH₂ 46 (1.0 mg, 3.8μmol, 1.3 eq.). The mixture was shaken for 10 min at room temperatureand then acidified with TFA (2 μL). RP-HPLC (3 mL/min, 0% to 90% B in 32min) gave 47 (1.5 mg, 77%) as a green solid.

¹H NMR (600 MHz, DMSO-d₆) δ=0.35 (br. s, 8H (should be 6H)), 2.24 (t,J=7.4 Hz, 2H), 2.55-2.65 (m, 1H, overlaps with the residual solventsignal), 2.92 (s, 6H), 2.94 (s, 6H), 4.23 (d, J=5.9 Hz, 2H), 5.48 (s,2H), 5.66 (t, J=7.0 Hz, 1H), 6.80-6.87 (m, 2H), 6.97-7.05 (m, 2H), 7.22(d, J=7.9Hz, 2H), 7.26 (d, J=8.5 Hz, 1H), 7.29 (d, J=8.5 Hz, 1H), 7.39(d, J=8.0 Hz, 2H), 8.35 (t, J=6.0 Hz, 1H), 8.94 ppm (s, 1H); HRMS (ESI):m/z calcd for C₃₆H₄₃N₈O₂Si⁺ [M+H]⁺ 647.3278, found 647.3274; LCMS (LC,10% to 100%): t_(R)=2.41 min.

N-(4-((4R,7R,10S,13S,19S,E)-7-((1H-Indol-2-yl)methyl)-4-(4-hydroxyphenyl)-8,13,15,19-tetramethyl-2,6,9,12-tetraoxo-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl)butyl)-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide(49, PA-SiR-Actin)

A solution of 2a (1.8 mg, 4.5 μmol, 1.0 eq.) in DMSO (300 μL) wastreated with DIEA (2.2 μL, 13.5 μmol, 3.0 eq.) and TSTU (1.6 mg, 5.4μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature.In a separate vial a solution of jasplakinolide-NHBoc 48 (3.8 mg, 5.0μmol, 1.1 eq.) in CH₂Cl₂/TFA (8:2, 80 μL) was shaken for 2 min. Thesolution was evaporated and dried on the high vacuum for 1 h. Theresidue was taken up in DMSO (50 μL) and added to the other mixture. Themixture was shaken for 30 min and then acidified with TFA (3 μL).RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 49 (1.3 mg, 27%) as alight blue solid.

¹H NMR (400 MHz, DMSO-d₆) δ=0.37 (s, 6H), 0.73-0.83 (m, 4H), 0.89 (d,J=6.8 Hz, 3H), 1.06 (p, J=7.1, 6.7 Hz, 1H), 1.15 (d, J=6.3 Hz, 3H),1.19-1.29 (m, 6H), 1.30-1.41 (m, 1H), 1.42-1.55 (m, 5H), 1.75-1.91 (m,2H), 2.08-2.20 (m, 3H), 2.58-2.71 (m, 2H), 2.72-2.93 (m, 2H), 2.93-2.96(m, 12H), 3.00 (s, 3H), 4.44-4.58 (m, 1H), 4.66 (h, J=6.3 Hz, 1H), 4.91(t, J=7.1 Hz, 1H), 5.18 (ddd, J=11.6, 8.8, 3.1 Hz, 1H), 5.50 (dd,J=11.3, 5.2 Hz, 1H), 5.65 (t, J=7.1 Hz, 1H), 6.64-6.72 (m, 2H),6.77-6.88 (m, 2H), 6.93 (t, J=7.4 Hz, 1H), 6.97-7.06 (m, 3H), 7.09-7.15(m, 2H), 7.23-7.34 (m, 3H), 7.66 (t, J=7.7 Hz, 2H), 7.73 (t, J=5.6 Hz,1H), 8.64 (d, J=8.8 Hz, 1H), 9.30 (br. s, 1H), 10.78 ppm (d, J=2.3 Hz,1H); HRMS (ESI): m/z calcd for C₆₁H₈₀N₇O₇Si⁺ [M+H]⁺ 1050.5883, found1050.5902; LCMS (LC, 10% to 90%): t_(R)=3.91 min.

4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(4-(4-(6-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)henoxy)butyl)butanamide(51, PA-SiR-DNA)

A solution of 2a (1.1 mg, 3.0 μmol, 1.0 eq.) in DMSO (150 μL) wastreated with DIEA (1.5 μL, 9.0 μmol, 3.0 eq.) and HATU (1.4 mg, 3.6μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature.In a separate vial a solution of 50 (2.1 mg, 3.6 μmol, 1.2 eq.) inCH₂Cl₂/TFA (8:2, 80 μL) was shaken for 5 min. The solution wasevaporated and dried on the high vacuum for 1 h. The residue was takenup in DMSO (50 μL) and added to the other mixture. The mixture wasshaken for 15 min and then acidified with TFA (3 μL) and diluted withwater (200 μL). RP-HPLC (3 mL/min, 0% to 90% B in 32 min) gave 51 (1.4mg, 53%) as a light green solid.

¹H NMR (400 MHz, CD₃OD) δ=0.49 (d, J=7.8 Hz, 6H), 1.59-1.75 (m, 2H),1.75-1.89 (m, 2H), 2.36 (t, J=7.3 Hz, 2H), 2.71 (q, J=7.3 Hz, 2H), 3.02(s, 3H), 3.15 (s, 6H), 3.17 (s, 6H), 3.25 (t, J=6.9 Hz, 2H), 3.61-3.77(m, 4H), 3.90-4.03 (m, 4H), 4.07 (t, J=6.2 Hz, 2H), 5.93 (t, J=7.2 Hz,1H), 7.14 (d, J=8.7 Hz, 2H), 7.23 (dd, J=8.5, 2.7 Hz, 1H), 7.29-7.35 (m,2H), 7.38-7.44 (m, 2H), 7.45-7.52 (m, 2H), 7.58 (d, J=8.5 Hz, 1H), 7.73(d, J=9.0 Hz, 1H), 7.90 (d, J=8.6 Hz, 1H), 8.04 (dd, J=8.6, 1.7 Hz, 1H),8.11 (d, J=8.6 Hz, 2H), 8.40 ppm (d, J=1.7 Hz, 1H); HRMS (ESI): m/zcalcd for C₅₂H₆₂N₉O₂Si⁺ [M+H]⁺ 872.4790, found 872.4797; LCMS (LC, 10%to 100%): t_(R)=2.42 min.

(2αR,4S,4αS,6R,9S,11S,12S,12αR,12βS)-12β-Acetoxy-9-((3-(4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-4α,8,13,13-tetramethyl-5-oxo-2α,3,4,4α,5,6,9,10,11,12,12α,12β-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-β]oxet-12-ylbenzoate (53)

A solution of 2a (1.8 mg, 4.6 μmol, 1.0 eq.) in DMSO (130 μL) wastreated with DIEA (2.3 μL, 13.8 μmol, 3.0 eq.) and TSTU (1.7 mg, 5.5μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature.In a separate vial a solution of docetaxel (52) (20 mg, 25.2 μmol, 5.5eq.) in formic acid (370 μL) was shaken for 45 min. The solution wasevaporated, coevaporated with toluene (3×500 μL) and dried on the highvacuum for 1 h. The residue was taken up in DMSO (200 μL) and a quarterof this solution (50 μL, 6.3 μmol, 1.4 eq.) was added to the othermixture. The mixture was shaken for 1 h and then acidified with TFA (3μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 53 (0.47 mg, 9%) asa light blue solid.

HRMS (ESI): m/z calcd for C₆₁H₇₄N₃O₁₃Si⁺ [M+H]⁺ 1084.4985, found1084.5005; LCMS (LC, 10% to 90%): t_(R)=2.74 min.

8-(4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)octanoicacid (55)

A solution of 2a (1.6 mg, 4.0 μmol, 1.0 eq.) in DMSO (120 μL) wastreated with DIEA (4.6 μL, 28.0 μmol, 7.0 eq.) and TSTU (1.4 mg, 4.8μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature.Amino-octanoic acid (54) (1.5 mg, 9.2 μmol, 2.3 eq.) was added to themixture. The mixture was shaken for 10 min and then acidified with TFA(3 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 55 (1.0 mg, 47%)as a blue solid.

HRMS (ESI): tn/z calcd for C₃₁H₄₆N₃O₃Si⁺ [M+H]⁺ 536.3303, found536.3293; LCMS (LC, 10% to 90%): t_(R)=2.92 min.

(2αR,4S,4αS,6R,9S,11S,12S,12αR,12βS)-12β-Acetoxy-9-((3-(8-(4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)octanamido)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-4α,8,13,13-tetramethyl-5-oxo-2α,3,4,4α,5,6,9,10,11,12,12α,12β-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-β]oxet-12-ylbenzoate (56, PA-SiR-Tubulin)

A solution of 55 (1.0 mg, 2.0 μmol, 1.0 eq.) in DMSO (120 ∞L) wastreated with DIEA (1.0 μL, 6.0 μmol, 3.0 eq.) and TSTU (0.7 mg, 2.4μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature.In a separate vial a solution of docetaxel (52) (20 mg, 25.2 μmol, 12.6eq.) in formic acid (370 μL) was shaken for 45 min. The solution wasevaporated, coevaporated with toluene (3×500 μL) and dried on the highvacuum for 1 h. The residue was taken up in DMSO (200 μL) and a tenth ofthis solution (20 μL, 2.5 μmol, 1.3 eq.) was added to the other mixture.The combined mixture was shaken for 1 h and then acidified with TFA (3μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 56 (0.24 mg, 10%)as a blue solid.

HRMS (ESI): tn/z calcd for C₆₉H₈₉N₄O₁₄Si⁺ [M+H]⁺ 1225.6139, found1225.6103; LCMS (LC): t_(R)=3.72 min, 10% to 90% gradient.

4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)butanamide(58)

A solution of 2a (1.0 mg, 2.5 μmol, 1.0 eq.) in DMSO (120 μL) wastreated with DIEA (2.1 μL, 12.5 μmol, 5.0 eq.) and TSTU (0.9 mg, 3.0μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature.Me-Tetrazine-NH₂.HCl (57) (0.7 mg, 3.0 μmol, 1.2 eq.) was added to themixture. The mixture was shaken for 10 min and then acidified with TFA(3 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 58 (0.70 mg,50%) as a purple solid.

¹H NMR (400 MHz, CD₃OD) δ=0.41 (s, 6H), 2.46 (t, J=6.9 Hz, 2H), 2.78 (q,J=7.0 Hz, 2H), 3.04 (s, 3H), 3.13 (s, 6H), 3.22 (s, 6H), 4.42-4.50 (m,2H), 5.96 (t, J=7.1 Hz, 1H), 7.17 (d, J=8.6 Hz, 1H), 7.35 (s, 2H),7.39-7.44 (m, 2H), 7.42-7.50 (m, 2H), 7.56 (d, J=8.5 Hz, 1H), 8.28-8.34(m, 2H), 8.60 ppm (t, J=6.0 Hz, 1H); HRMS (ESI): m/z calcd forC₃₃H₄₀N₇OSi⁺ [M+H]⁺ 578.3058, found 578.3051; LCMS (LC, 10% to 90%):t_(R)=3.31 min.

4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide(59)

A solution of 6a (1.3 mg, 3.0 μmol, 1.0 eq.) in DMSO (150 μL) wastreated with DIEA (1.5 μL, 9.0 μmol, 3.0 eq.) and TSTU (1.1 mg, 3.6μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature.In a separate vial a solution of Halo-NHBoc 43 (1.3 mg, 4.0 μmol, 1.3eq.) in CH₂Cl₂/TFA (8:2, 80 μL) was shaken for 3 min. The solution wasevaporated and dried on the high vacuum for 1 h. The residue was takenup in DMSO (50 μL) and added to the other mixture. The mixture wasshaken for 10 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min,10% to 90% B in 32 min) gave 59 (1.02 mg, 55%) as a blue oil.

¹H NMR (400 MHz, CD₃CN) δ=1.03 (d, J=7.4 Hz, 12H), 1.29-1.44 (m, 4H),1.48-1.57 (m, 4H), 1.69-1.76 (m, 2H), 2.19 (t, J=7.4 Hz, 2H), 2.51-2.63(m, 2H), 3.03 (s, 6H), 3.04 (s, 6H), 3.23-3.31 (m, 2H), 3.37 (d, J=6.5Hz, 2H), 3.42 (d, J=5.7 Hz, 2H), 3.48-3.53 (m, 4H), 3.56-3.61 (m, 2H),5.72 (t, J=7.2 Hz, 1H), 6.43 (s, 1H), 7.04 (dd, J=8.5, 2.7 Hz, 1H),7.14-7.22 (m, 1H), 7.26 (d, J=2.7 Hz, 1H), 7.33 (d, J=2.7 Hz, 1H), 7.41(d, J=8.5 Hz, 1H), 7.51 ppm (d, J=8.6 Hz, 1H); HRMS (ESI): m/z calcd forC₃₇H₅₉ClN₃O₃Si⁺ [M+H]⁺ 656.4009, found 656.4009; LCMS (LC, 10% to 90%):t_(R)=5.09 min.

(9H-Fluoren-9-yl)methyl(21-chloro-8-oxo-3,6,12,15-tetraoxa-9-azahenicosyl)carbamate(61)

A solution of Fmoc-N-PEG2-AcOH 60 (21.2 mg, 55.0 μmol, 1.1 eq.) in DMSO(625 μL) was treated with DIEA (41 μL, 250.0 μmol, 5.0 eq.) and TSTU(18.1 mg, 60.0 μmol, 1.2 eq.). The mixture was shaken for 20 min at roomtemperature. In a separate vial a solution of Halo-NHBoc 43 (16.2 mg,50.0 μmol, 1.0 eq.) in CH₂Cl₂/TFA (8:2, 750 μL) was shaken for 5 min.The solution was evaporated and dried on the high vacuum for 1 h. Theresidue was taken up in DMSO (100 μL) and added to the other mixture.The mixture was shaken for 10 min and then acidified with TFA (20 μL).RP-HPLC (4 mL/min, 10% to 90% B in 32 min) gave Halo-PEG2 61 (19 mg,63%) as a colourless oil.

¹H NMR (400 MHz, ACN-d₃) δ=1.23-1.34 (m, 2H), 1.34-1.42 (m, 2H),1.43-1.54 (m, 2H), 1.61-1.78 (m, 2H), 3.26 (q, J=5.5 Hz, 2H), 3.27-3.40(m, 4H), 3.39-3.47 (m, 4H), 3.46-3.52 (m, 4H), 3.52-3.64 (m, 6H), 3.89(s, 2H), 4.22 (t, J=6.7 Hz, 1H), 4.37 (d, J=6.6 Hz, 2H), 5.90 (s, 1H),7.16 (s, 1H), 7.33 (t, J=7.5 Hz, 2H), 7.41 (t, J=7.4 Hz, 2H), 7.65 (d,J=7.4 Hz, 2H), 7.83 ppm (d, J=7.5 Hz, 2H); ¹³C NMR (101 MHz, ACN-d₃)δ=26.10, 27.34, 30.23, 33.27, 39.19, 41.54, 46.17, 48.12, 66.69, 70.33,70.56, 70.61, 70.64, 70.88, 70.95, 71.53, 71.71, 120.93, 126.01, 128.05,128.60, 142.13, 145.22, 157.34, 170.79 ppm; HRMS (ESI): m/z calcd forC₃₁H₄₄ClN₂O₇ ⁺ [M+H]⁺ 591.2832, found 591.2831; LCMS (LC, 10% to 90%):t_(R)=3.42 min.

(9H-Fluoren-9-yl)methyl(28-chloro-15-oxo-3,6,9,12,19,22-hexaoxa-16-azaoctacosyl)carbamate(63)

A solution of Fmoc-N-PEG4-CH₂-AcOH 62 (27.0 mg, 55.0 μmol, 1.1 eq.) inDMSO (625 μL) was treated with DIEA (41 μL, 250.0 μmol, 5.0 eq.) andTSTU (18.1 mg, 60.0 μmol, 1.2 eq.). The mixture was shaken for 20 min atroom temperature. In a separate vial a solution of Halo-NHBoc 43 (16.2mg, 50.0 μmol, 1.0 eq.) in CH₂Cl₂/TFA (8:2, 750 μL) was shaken for 5min. The solution was evaporated and dried on the high vacuum for 1 h.The residue was taken up in DMSO (50 μL) and added to the other mixture.The mixture was shaken for 10 min and then acidified with TFA (20 μL).RP-HPLC (4 mL/min, 10% to 90% B in 32 min) gave Halo-PEG4 63 (19 mg,55%) as colourless oil.

¹H NMR (400 MHz, ACN-d₃) δ=1.27-1.45 (m, 4H), 1.46-1.58 (m, 2H),1.68-1.80 (m, 2H), 2.34 (t, J=6.1 Hz, 2H), 3.23 (q, J=5.5 Hz, 2H), 3.29(q, J=5.6 Hz, 2H), 3.39 (t, J=6.6 Hz, 2H), 3.44-3.58 (m, 22H), 3.63 (t,J=6.1 Hz, 2H), 4.23 (t, J=6.8 Hz, 1H), 4.35 (d, J=6.8 Hz, 2H), 5.80 (s,1H), 6.69 (s, 1H), 7.34 (td, J=7.4, 1.1 Hz, 2H), 7.42 (t, J=7.4 Hz, 2H),7.66 (d, J=7.5 Hz, 2H), 7.83 ppm (d, J=7.5 Hz, 2H); ¹³C NMR (101 MHz,ACN-d₃) δ=26.13, 27.34, 30.27, 33.28, 37.32, 39.85, 41.52, 46.18, 48.12,66.78, 67.82, 70.19, 70.37, 70.70, 70.91, 70.94, 70.97, 71.07, 71.13,71.57, 120.93, 126.06, 128.06, 128.62, 142.12, 145.21, 157.29, 171.97ppm; HRMS (ESI): m/z calcd for C₃₆H₅₄ClN₂O₉ ⁺ [M+H]⁺ 693.3512, found693.3514; LCMS (LC, 10% to 90%): t_(R)=4.19 min.

4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)-N-(21-chloro-8-oxo-3,6,12,15-tetraoxa-9-azahenicosyl)butanamide(64)

A solution of 6a (1.1 mg, 2.5 μmol, 1.0 eq.) in DMSO (120 μL) wastreated with DIEA (2 μL, 12.5 μmol, 5.0 eq.) and TSTU (0.9 mg, 3.0 μmol,1.2 eq.). The mixture was shaken for 20 min at room temperature. In aseparate vial a solution of Halo-PEG2 61 (1.8 mg, 3.0 μmol, 1.2 eq.) inACN/piperidine (9:1, 100 μL) was shaken for 15 min. The solution wasevaporated and dried on the high vacuum for 1 h. The residue was takenup in DMSO (50 μL) and added to the other mixture. The mixture wasshaken for 10 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min,10% to 100% B in 32 min) gave 64 (1.29 mg, 64%) as a blue oil.

HRMS (ESI): m/z calcd for C₄₃H₇₀ClN₄O₆Si⁺ [M+H]⁺ 801.4748, found801.4742; LCMS (LC, 10% to 90%): t_(R)=4.05 min.

1-(4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3,6,9,12-tetraoxapentadecan-15-amide(65)

A solution of 6a (1.1 mg, 2.5 μmol, 1.0 eq.) in DMSO (120 μL) wastreated with DIEA (2 μL, 12.5 μmol, 5.0 eq.) and TSTU (0.9 mg, 3.0 μmol,1.2 eq.). The mixture was shaken for 20 min at room temperature. In aseparate vial a solution of Halo-PEG4 63 (1.8 mg, 3.0 μmol, 1.2 eq.) inACN/piperidine (9:1, 100 μL) was shaken for 15 min. The solution wasevaporated and dried on the high vacuum for 1 h. The residue was takenup in DMSO (50 μL) and added to the other mixture. The mixture wasshaken for 10 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min,10% to 100% B in 32 min) gave 65 (0.48 mg, 21%) as a blue oil.

HRMS (ESI): m/z calcd for C₄₈H₈₀ClN₄O₈Si⁺ [M+H]⁺ 903.5428, found903.5424; LCMS (LC, 10% to 90%): t_(R)=4.37 min.

N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamide(66)

A solution of 6a (1.5 mg, 3.3 μmol, 1.0 eq.) in DMSO (150 μl) wastreated with DIEA (1.6 μL, 9.9 μmol, 3.0 eq.) and TSTU (1.2 mg, 4.0μmol, 1.2 eq.) was shaken for 20 min. BG-NH₂ 46 (1.1 mg, 4.0 μmol, 1.2eq.) was added. The mixture was shaken for 10 min at room temperatureand acidified with TFA (2 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32min) gave 66 (0.73 mg, 31%) as a light blue solid.

¹H NMR (400 MHz, DMSO-d₆) δ=0.99 (d, J=7.4 Hz, 12H), 1.37-1.53 (m, 2H),2.20 (t, J=7.5 Hz, 2H), 2.52-2.59 (m, 2H), 2.93 (s, 6H), 2.95 (s, 6H),4.23 (d, J=5.7 Hz, 2H), 5.50 (s, 2H), 5.59 (t, J=7.0 Hz, 1H), 6.85 (d,J=8.6 Hz, 1H), 6.90 (d, J=8.2 Hz, 1H), 6.99 (s, 2H), 7.21-7.32 (m, 3H),7.35 (d, J=8.6 Hz, 1H), 7.45 (d, J=7.8 Hz, 2H), 8.38 (t, J=5.9 Hz, 1H),8.43-8.47 ppm (m, 1H); HRMS (ESI): m/z calcd for C₄₀H₅₁N₈O₂Si⁺ [M+H]⁺703.3899, found 703.3901; LCMS (LC, 10% to 90%): t_(R)=3.44 min.

2-Amino-3-((4-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)amino)-3-oxopropane-1-sulfonicacid (68)

A solution of Fmoc-cysteic-acid (67) (8.6 mg, 22.0 μmol, 1.1 eq.) inDMSO (250 μl) was treated with DIEA (165 μL, 100.0 μmol, 5.0 eq.) andTSTU (7.2 mg, 24.0 μmol, 1.2 eq.) was shaken for 20 min. BG-NH₂ 46 (5.4mg, 20.0 μmol, 1.0 eq.) was added and the mixture was shaken for 45 minat room temperature. The mixture was poured into Et₂O (2 mL) and theprecipitate was washed with Et₂O (2×1 mL). The solid was redissolved inACN/DBU 9:1 (500 μL) and shaken for 10 min. The mixture was acidifiedwith TFA (2 μL) and diluted with water and DMSO. RP-HPLC (4 mL/min, 10%to 70% B in 32 min) gave 68 (5 mg, 62%) as a light blue solid.

HRMS (ESI): m/z calcd for C₁₆H₂₀N₇O₅S⁺ [M+H]⁺ 422.1241, found 422.1241;LCMS (LC, 10% to 90%): t_(R)=0.73 min.

3-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)amino)-2-(4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-3-oxopropane-1-sulfonicacid (69)

A solution of 6a (1.1 mg, 2.5 μmol, 1.0 eq.) in DMSO (120 μl) wastreated with DIEA (24 μL, 150.0 μmol, 60.0 eq.) and TSTU (0.9 mg, 3.0μmol, 1.2 eq.) was shaken for 10 min. 68 (1.2 mg, 3.0 μmol, 1.2 eq.) wasadded. The mixture was shaken for 10 min at room temperature and thenacidified with TFA (20 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min)gave 69 (0.27 mg, 13%) as a light blue solid.

HRMS (ESI): m/z calcd for C₄₃H₅₆N₉O₆SSi⁺ [M+H]⁺ 854.3838, found854.3823; LCMS (LC, 10% to 90%): t_(R)=2.64 min.

N-(4-((4R,7R,10S,13S,19S,E)-7-((1H-Indol-2-yl)methyl)-4-(4-hydroxyphenyl)-8,13,15,19-tetramethyl-2,6,9,12-tetraoxo-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl)butyl)-4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamide(70)

A solution of 6a (1.3 mg, 3.0 μmol, 1.0 eq.) in DMSO (150 μL) wastreated with DIEA (1.5 μL, 9.0 μmol, 3.0 eq.) and TSTU (1.1 mg, 3.6μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature.In a separate vial a solution of jasplakinolide-NHBoc 48 (2.8 mg, 3.6μmol, 1.2 eq.) in CH₂Cl₂/TFA (8:2, 80 μL) was shaken for 3 min. Thesolution was evaporated and dried on the high vacuum for 1 h. Theresidue was taken up in DMSO (50 μL) and added to the other mixture. Themixture was shaken for 10 min and then acidified with TFA (3 μL).RP-HPLC (3 mL/min, 0% to 90% B in 32 min) gave 70 (0.75 mg, 23%) as alight blue solid.

¹H NMR (400 MHz, CD₃CN) δ=0.95-1.02 (m, 4H), 1.00-1.09 (m, 13H), 1.11(d, J=6.3 Hz, 4H), 1.27-1.39 (m, 4H), 1.48-1.62 (m, 2H), 1.78-1.89 (m,5H), 2.20-2.34 (m, 4H), 2.40-2.52 (m, 2H), 2.62-2.71 (m, 3H), 2.71-2.88(m, 2H), 2.95 (s, 3H), 3.04 (s, 6H), 3.09 (s, 6H), 3.20 (s, 3H), 4.69(q, J=5.9 Hz, 1H), 4.74-4.80 (m, 1H), 4.96 (t, J=6.9 Hz, 1H), 5.10-5.19(m, 1H), 5.54 (dt, J=10.1, 5.0 Hz, 1H), 5.82 (t, J=7.3 Hz, 1H), 6.59 (d,J=6.9 Hz, 2H), 6.76 (d, J=6.5 Hz, 2H), 6.98-7.05 (m, 4H), 7.09-7.11 (m,2H), 7.28-7.40 (m, 4H), 7.44-7.54 (m, 2H), 7.59 (dd, J=13.1, 8.2 Hz,2H), 8.35-8.53 (m, 1H), 10.11 ppm (s, 1H); HRMS (ESI): m/z calcd forC₆₅H₈₈N₇O₇Si⁺ [M+H]⁺ 1106.6509, found 1106.6520; LCMS (LC, 10% to 90%):t_(R)=4.86 min.

N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-(3,7-bis(dimethylamino)-2,8-difluoro-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide(71)

A solution of 36 (1.2 mg, 2.7 μmol, 1.0 eq.) in DMSO (120 μl) wastreated with DIEA (1.3 μL, 8.1 pmol, 3.0 eq.) and TSTU (1.0 mg, 3.2∥mol, 1.2 eq.) was shaken for 20 min. BG-NH₂ 46 (0.9 mg, 3.2 μmol, 1.2eq.) was added. The mixture was shaken for 10 min at room temperatureand then acidified with TFA (2 μL). RP-HPLC (3 mL/min, 10% to 90% B in32 min) gave 71 (1.1 mg, 60%) as a white solid.

¹H NMR (400 MHz, CD₃OD) δ=0.40 (s, 6H), 2.35-2.45 (m, 2H), 2.72 (t,J=7.1 Hz, 2H), 2.93 (s, 6H), 2.94 (s, 6H), 4.37 (d, J=4.3 Hz, 2H), 5.60(s, 2H), 5.87 (t, J=7.2 Hz, 1H), 7.18 (d, J=9.8 Hz, 1H), 7.22 (d, J=10.0Hz, 1H), 7.25-7.33 (m, 4H), 7.34-7.42 (m, 2H), 8.34 (s, 1H), 8.52 ppm(t, J=6.0 Hz, 1H); ¹⁹F NMR (376 MHz, CD₃OD) δ=−123.44-−123.20 (m),−123.13-−122.77 ppm (m); HRMS (ESI): m/z calcd for C₃₆H₄₁F₂N₈O₂Si⁺[M+H]⁺ 683.3084, found 683.3082; LCMS (LC, 10% to 90%): t_(R)=3.50 min.

N-(4-((4R,7R,10S,13S,19S,E)-7-((1H-Indol-2-yl)methyl)-4-(4-hydroxyphenyl)-8,13,15,19-tetramethyl-2,6,9,12-tetraoxo-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl)butyl)-4-(3,7-bis(dimethylamino)-2,8-difluoro-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide(72)

A solution of 36 (1.3 mg, 3.0 μmol, 1.0 eq.) in DMSO (120 μL) wastreated with DIEA (1.5 μL, 9.0 μmol, 3.0 eq.) and TSTU (1.1 mg, 3.6μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature.In a separate vial a solution of jasplakinolide-NHBoc 48 (2.8 mg, 3.6μmol, 1.2 eq.) in CH₂Cl₂/TFA (8:2, 80 μL) was shaken for 2 min. Thesolution was evaporated and dried on the high vacuum for 1 h. Theresidue was taken up in DMSO (50 μL) and added to the other mixture. Themixture was shaken for 10 min and then acidified with TFA (3 μL).RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 72 (0.22 mg, 6%) as awhite solid.

¹H NMR (400 MHz, CD₃OD) δ=0.46 (s, 6H), 0.73-1.01 (m, 4H), 1.05 (d,J=6.8 Hz, 3H), 1.16 (d, J=6.3 Hz, 3H), 1.18-1.26 (m, 1H), 1.29 (s, 3H),1.29-1.47 (m, 2H), 1.50-1.71 (m, 5H), 1.81-1.94 (m, 3H), 2.34 (q, J=7.0Hz, 2H), 2.59 (ddd, J=10.4, 6.9, 3.0 Hz, 1H), 2.70 (dd, J=8.9, 5.1 Hz,3H), 2.80-3.01 (m, 2H), 3.03 (d, J=1.2 Hz, 9H), 3.08 (s, 6H), 4.70 (t,J=5.7 Hz, 1H), 4.78-4.84 (m, 1H), 5.02 (t, J=7.0 Hz, 1H), 5.24 (td,J=9.1, 4.0 Hz, 1H), 5.56-5.66 (m, 1H), 5.93 (t, J=7.3 Hz, 1H), 6.69-6.75(m, 2H), 6.93-7.03 (m, 3H), 7.03-7.09 (m, 2H), 7.24-7.39 (m, 3H), 7.46(dd, J=11.2, 9.3 Hz, 2H), 7.56-7.63 (m, 1H), 8.39 ppm (d, J=8.5 Hz, 1H);HRMS (ESI): m/z calcd for C₆₁H₇₈F₂N₇O₇Si⁺ [M+H]⁺ 1086.5695, found1086.5704; LCMS (LC, 10% to 90%): t_(R)=4.85 min.

Abbreviations

ACN, acetonitrile; NBS, N-bromosuccinimide; PBS, phosphate bufferedsaline; secBuLi secondary butyl lithium;

1. A compound characterized by general formula (100):

wherein R¹ and R⁶ are independently selected from hydrogen and fluorine,particularly R¹ and R⁶ are hydrogen; R², R³, R⁴ and R⁵ independently ofeach other is selected from H, halogen, SO₃H, CO₂H, NO₂, CO₂R, SO₂R(with R being selected from C₁ to C₄ unsubstituted alkyl) and anunsubstituted or substituted (particularly unsubstituted or halogen-,amino-, hydroxyl-, SO₃H- and/or carboxyl substituted) moiety selectedfrom C₁-C₂₀ alkyl, C₃-C₈ cycloalkyl, C₁-C₂₀ alkoxy, C₂-C₂₀ alkenyl,C₂-C₂₀ alkynyl, C₇-C₂₀ alkylaryl, phenyl and 5- or 6-membered ringheteroaryl, or a combination thereof; R⁷ and R⁸ are independentlyselected from: unsubstituted and substituted C₁-C₁₂ alkyl, C₃-C₈cycloalkyl, C₂-C₁₂ alkylene, C₂-C₁₂ alkylyne and C₇-C₁₂ alkylaryl;unsubstituted or substituted 5- or 6-ring aryl, particularly phenyl,particularly wherein R⁷ is selected from H and a substituted orunsubstituted alkyl, alkenyl, alkenyl, alkylary or aryl having x carbonatoms and R⁸ is selected from H and a substituted or unsubstitutedalkyl, alkenyl, alkenyl, alkylary or aryl having y carbon atoms and thesum of x and y is between 0 and 12, more particularly between 0 and 10,even more particularly between 0 and 8, even more particularly between 0and 6; R^(N1), R^(N2), R^(N3) and R^(N4) are: independently selectedfrom H, unsubstituted and substituted C₁-C₈ alkyl, C₃-C₈ cycloalkyl,C₁-C₄ acyl, and C₇-C₁₂ alkylaryl, and unsubstituted phenyl or phenylsubstituted by COOH—, COOR, CONR₂, unsubstituted alkyl, halogen,O-alkyl, and/or NO₂; or R^(N1) together with R^(N2), and/or R^(N3)together with R^(N4) are a C₃, C₄, C₆ unsubstituted or substituted alkylforming a 3-7 sized ring structure; or R^(N1) and/or R^(N3) areindependently selected from H and unsubstituted and substituted C₁-C₈alkyl, C₃-C₈ cycloalkyl, and C₇-C₁₂ alkylaryl, and R^(N2) together withR² or R³, and/or R^(N4) together with R⁴ or R⁵, is an unsubstituted orsubstituted C₂, C₃ or C₄ alkyl, or an unsubstituted or substituted C₂,C₃ or C₄ N-, O-, S-, or Se-alkyl; one of R⁹ and R¹⁹ is hydrogen and theother one is hydrogen or a saturated carbon atom connected to a moietyselected from H, halogen, SO₃H, CO₂H, NO₂, CO₂R, SO₂R (with R beingselected from C₁ to C₄ unsubstituted alkyl) and an unsubstituted orsubstituted (particularly unsubstituted or halogen-, amino-, hydroxyl-,SO₃H— and/or carboxyl substituted) moiety selected from C₁-C₂₀ alkyl,C₃-C₈ cycloalkyl, C₁-C₂₀ alkoxy, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, C₇-C₂₀alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combinationthereof and wherein optionally the compound is covalently linked to abinding moiety M via any of the substituents.
 2. The compound accordingto claim 1, wherein the compound is covalently linked (particularlythrough any one of substituents R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R^(N1), R^(N2), R^(N3) and R^(N4)) to a binding moiety M selected from:a. a moiety selectively attachable by covalent bond to a protein ornucleic acid under conditions prevailing in cell culture or inside of aliving cell, particularly a moiety able to form an ester bond, an etherbond, an amide bond, a disulfide bond, a Schiff base, or a moiety ableto react in a click-chemistry reaction, more particularly selected from—COCHCH₂, —CO—NHS, biotin, an azide or ethyne moiety, a tetrazinemoiety, a (bicyclo[6.1.0]nonyne) moiety, a cyclooctyne moiety, atranscyclooctene moiety and a maleimide, or from b. a substrate ofO⁶-alkylguanine-DNA-alkyltransferase, particularly a6-[(4-methylenephenyl)methoxy]-9H-purin-2-amine moiety of formula (110),or a pyrimidine derivative thereof, particularly a moiety of formula(111) or (112),

c. a substrate of a haloalkane halotransferase, particularly a1-chlorohexyl moiety as exemplarily shown below;

or from d. a substrate of dihydrofolate reductase, particularly themoiety:

e. a moiety capable of selectively interacting non-covalently with abiomolecule (particularly a protein or nucleic acid) under conditionsprevailing in a live cell, wherein said moiety and said biomolecule forma complex having a dissociation constant k_(D) of 10⁻⁶ mold or less,particularly wherein M has a molecular mass of more than 160 u but lessthan 1000 u, particularly less than 700 u, more particularly less than500 u, and M comprises up to five hydrogen bond donators, up to tenhydrogen bond acceptors and is characterized by an octanol-waterpartition coefficient logP of below 5.6; more particularly, M isselected from taxol, jasplaklinolide, a bis-benzimide DNA stain,pepstatin A and triphenylphosphonium; or wherein M is an oligonucleotidehaving a sequence length of 10 to 40 nucleotides. f. a lipid,particularly a lipid selected from a ceramide derivative, a glyceride,or a fatty acid.
 3. The compound according to claim 1, wherein any oneof substituents R², R³, R⁴, R⁵, and one of R⁹ and R¹⁰ independently ofany other is H or a moiety having a molecular weight between 15 and 1500u (g/mol); particularly wherein one of substituents R², R³, R⁴, R⁵, R⁹and R¹⁰ is a moiety having a molecular weight between 15 and 1500 u andthe other ones are selected from H and unsubstituted or fluoro-, amino-,hydroxyl-, SO₃H— and/or carboxyl substituted C₁ to C₄ alkyl, alkenyl oralkynyl; more particularly wherein one of substituents R², R³, R⁴, R⁵,R⁹ and R¹⁹ is a moiety having a molecular weight between 15 and 1500 uand the other ones are H.
 4. The compound according to claim 3, whereinsaid moiety having a molecular weight between 15 and 1500 u ischaracterized by a general formula -L-M, wherein L is a linkercovalently connecting the compound of structure (1) to the bindingmoiety M as defined above, and L is a covalent bond or a linkerconsisting of 1 to 50 atoms having an atomic weight of 12 or higher,particularly wherein said moiety having a molecular weight between 15and 1500 u is characterized by a general formulaL^(A1) _(n)-L^(J1) _(n)′-L^(A2) _(m)-L^(J2) _(m)′-L^(A3) _(p)-L^(J3)_(p′)-L^(A4) _(q)-L^(J4) _(q)′-M_(s), wherein L^(A1), L^(A2), L^(A3) andL^(A4) independently of each other are selected from C₁ to C₁₂unsubstituted or amino-, hydroxyl-, carboxyl- or fluoro substitutedalkyl or cycloalkyl, (CH₂—CH₂—O)_(r) or (CH₂—CH(OH)—CH₂—O)_(r) with rbeing an integer from 1 to 20, alkylaryl, alkylaryl-alkyl, andunsubstituted or alkyl-, halogen-, amino-, alkylamino-, imido-, nitro-,hydroxyl- oxyalkyl-, carbonyl-, carboxyl-, sulfuryl- and/or sulfoxylsubstituted aryl or heteroaryl, L^(J1), L^(J2), L^(J3) and L^(J4)independently of each other are selected from —NR⁵C(O)—, —C(O)N(R⁵)—,—CN—, —NC—, —CO—, —OC(O)—, —C(O)O—, —NR^(N5)—, —O—, —P(OOH)—, —OP(OOH)—,—P(OOH)O—, —OP(OOH)O—, —OP(OOH)O—, —S—, —SO—, SO₂—, with R^(N5) selectedfrom H and unsubstituted or amino-, hydroxyl-, carboxyl or fluorosubstituted C₁ to C₆ alkyl, particularly R^(N5) is selected from H andunsubstituted C₁ to C₃ alkyl; n, n′, m, m′, p, p′, q, q′ and sindependently from each other are selected from 0 and 1, and M has themeaning defined above.
 5. The compound according to claim 4, wherein Lis -L^(A1)-L^(J1)-L^(A2) _(m)-L^(J2) _(m)′-L^(A3) _(p), wherein L^(A1),L^(A2) and L^(A3) are independently selected from C₁ to C₆unsubstituted, amino-, hydroxyl-, carboxyl- or fluoro substituted alkylor cycloalkyl, and (CH₂—CH₂—O)_(r) or (CH₂—CH(OH)—CH₂—O)_(r) with rbeing an integer from 1 to 4, and L^(J1) and L^(J2) are selectedindependently from —NR⁵C(O)—, —C(O)N(R⁵)—, —CN——NC—, —CO—, —OC(O)—,—C(O)O—, NR^(N5)—, —O—, and —S—, and m, m′ and p independently from eachother are selected from 0 and
 1. 6. The compound according to claim 1,wherein R⁷ and R⁸ are independently selected from unsubstituted orhydroxyl-, amino- or halogen-substituted C₁ to C₄ alkyl, alkenyl oralkynyl, unsubstituted or hydroxyl-, amino- or halogen-substituted C₃ toC₆ cycloalkyl or unsubstituted or hydroxyl-, alkyoxy-, amino- orhalogen-substituted phenyl, particularly methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl, and phenyl, more particularlywherein R⁷ and R⁸ are the same.
 7. The compound according to claim 1,wherein a. R^(N1) and R^(N2), and/or R^(N3) and R^(N4), areindependently selected from H, unsubstituted and amino-, hydroxy-,carboxy- and/or fluoro-substituted C₁-C₆ alkyl, C₁-C₄ acyl, and C₃-C₆cycloalkyl, particularly R^(N1) and R^(N2), and/or R^(N3) and R^(N4),are independently selected from H, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, tert-butyl and CH₂CF₃, b. R^(N1) together withR^(N2), and/or R^(N3) together with R^(N4) together are an unsubstitutedor alkyl-, amino-, hydroxy-, carboxy- and/or fluoro-substituted C₃-C₆alkyl, particularly —(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅—, —(CH₂)₂O(CH₂)₂— or—(CH₂)₂NR^(NN)(CH₂)₂— with R^(NN) being selected from H andunsubstituted C₁ to C₄ alkyl; c. R^(N1) and/or R^(N3) are independentlyselected from H, unsubstituted and alkyl- (particularly methyl-),amino-, hydroxy-, carboxy- and/or fluoro-substituted C₁-C₆ alkyl, C₁-C₄acyl, and C₃-C₆ cycloalkyl, and R^(N2) together with R² or R³, and/orR^(N4) together with R⁴ or R⁵, is an alkyl or heteroalkyl bridgeselected from —(CH₂)₂—, —(CH₂)₃—, —CH₂CH═CH— or —(CH₂)₄— or —CH₂—O—,—CH₂—NR⁵—, —CH₂—S—, —CH₂—Se—, —(CH₂)₂O—, —(CH₂)₂NR^(N)—, —(CH₂)₂S—,—(CH₂)₂Se—, —CH₂—O—CH₂—, —CH₂NR⁵—, —CH₂S—CH₂—, —CH₂—Se—CH₂—,—CH₂-(1,2)phenyl-, and a mono- or dimethyl substituted derivative of anyone of the foregoing alkyl or heteroalkyl bridge moieties; d. R^(N1)and/or R^(N3) are independently selected from H, unsubstituted andalkyl- (particularly methyl-), amino-, hydroxy-, carboxy- and/orfluoro-substituted C₁-C₆ alkyl, C₁-C₄ acyl, and C₃-C₆ cycloalkyl, andR^(N2) together with R², and/or R^(N4) together with R⁵, form an annularstructure according to any one of substructures (101) to (104) or (101′)to (104′):

wherein R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are selected from H,unsubstituted or hydroxyl-, amino-, carboxyl-, sulfoxyl- orhalogen-substituted C₁ to C₄ alkyl, halogen, SO₃R, COOR′, CONR′₂ with Rselected from H and unsubstituted C₁ to C₄ alkyl; and R¹⁷ is selectedfrom H unsubstituted or hydroxyl-, amino-, carboxyl-, sulfoxyl- orhalogen-substituted C₁ to C₄ alkyl, halogen, NO₂, CN, SO₃R, COOR′,CONR+₂ with R selected from H and unsubstituted C₁ to C₄ alkyl;particularly wherein R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are selected fromH, methyl, CH₂—SO₃H, Cl and F, and R¹, R³, R⁷ and R⁵ can have any of themeanings given herein; or e. R^(N1) together with R³, and R^(N2)together with R², and/or R^(N3) together with R⁴, and R^(N4) togetherwith R⁵, form a bi-annular structure according to any one ofsubstructures (105) to (107) and/or (105′) to (107′):

wherein R¹¹, R¹², R¹³, and R¹⁵ are selected from H, unsubstituted orhydroxyl-, amino-, carboxyl-, sulfoxyl- or halogen-substituted C₁ to C₄alkyl, halogen, SO₃R, COOR′, CONR′₂ with R selected from H andunsubstituted C₁ to C₄ alkyl; particularly wherein R¹¹, R¹², R¹³, andR¹⁵ are selected from H, methyl, CH₂—SO₃H, Cl and F, and R¹, R³, R⁷ andR⁸ can have any of the meanings given herein; or f. R^(N2) and/or R^(N4)are independently selected from H, unsubstituted and alkyl-(particularly methyl-), amino-, hydroxy-, carboxy- and/orfluoro-substituted C₁-C₆ alkyl, C₁-C₄ acyl, and C₃-C₆ cycloalkyl, andR^(N1) together with R³, and/or R^(N3) together with R⁴, form an annularstructure according to any one of substructures (108) to (109) and/or(108′) to (109′):

wherein R¹, R³, R⁴, R⁶, R⁷ and R⁸ can have any of the meanings givenherein.
 8. The compound according to claim 7, wherein R^(N1) togetherwith R^(N2), and/or R^(N3) together with R^(N4) together are —(CH₂)₃—,—CH₂CHFCH₂—, —CH₂CF₂CH₂—, —CH₂CH(CH₃)CH₂—, —CH₂C(CH₃)₂CH₂—,CH₂CH(CN)CH₂—, CH₂CH(COOH)CH₂—, CH₂CH(CH₂COOH)CH₂—, —CH₂CH(OCH₃)CH₂— and—CH₂CH(N(CH₃)₂)CH₂—, particularly wherein the substituent is the samefor R^(N1) with R^(N2), and R^(N3) with R^(N4).
 9. The compoundaccording to claim 1, wherein R¹, R⁶ and R⁹ are H, and/or R², R³, R⁴ andR⁵ are selected from H, halogen, SO₃H, and unsubstituted and amino-,hydroxy-, carboxy-, SO₃H—, and/or halogen-substituted C₁-C₄ alkyl, CO₂H,CO₂R, SO₂R with R being selected from C₁ to C₄ unsubstituted alkyl,and/or R⁷ and R⁸ are independently selected from unsubstituted orhalogen-substituted C₁ to C₄ alkyl or C₃ to C₆ cycloalkyl and phenyl,and/or R^(N1), R^(N2), R^(N3) and R^(N4) are individually unsubstitutedor amino-, hydroxyl- or halogen-substituted C₁ to C₄ alkyl or C₃ to C₆cycloalkyl, or R^(N1) together with R^(N2), and R^(N3) together withR^(N4) together with the N form an unsubstituted or methyl-, ethyl-propyl-, or halogen-substituted aziridine, pyrrolidine, piperidine,piperazine or morpholine and/or R¹⁰ is selected from unsubstituted oramino-, hydroxyl-, carboxyl- and/or halogen-substituted C₂ to C₁₂ alkylor C₃ to C₇ cycloalkyl; -L^(A1) _(n)-L^(J1) _(n)′-L^(A2) _(m)-L^(J2)_(m)′-L^(A3) _(p)-L^(J3) _(p)′-L^(A4) _(q)-L^(J4) _(q)′-M_(s),whereinL^(A1 . . . 4), L^(J1 . . . 4), n, n′ . . . q′, s and M have thedefinitions recited above.
 10. The compound according to claim 1,wherein R² and R⁵ are F or Cl.
 11. The compound according to claim 1,wherein R¹, R², R³, R⁴, R⁵, R⁶ and R⁹ are H, R⁷ and R⁸ are C₁ to C₄alkyl or phenyl, R^(N1), R^(N2), R^(N3) and R^(N4) are individuallyunsubstituted or amino-, hydroxyl- or fluoro substituted C₁ to C₄ alkyl,or R^(N1) together with R^(N2), and R^(N3) together with R^(N4) togetherare —(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅—, —(CH₂)₂O(CH₂)₂— or —(CH₂)₂NH(CH₂)₂—and R¹⁹ is selected from unsubstituted or amino-, hydroxyl-, carboxyl-and/or fluoro substituted C₂ to C₁₂ alkyl or C₃ to C₇ cycloalkyl; or R¹⁰is -L^(A1) _(n)-L^(J1) _(n)′-L^(A2) _(m)-L^(J2) _(m)′-L^(A3) _(p)-L^(J3)_(p)′-L^(A4) _(q)-L^(J4) _(q)′-M_(s), wherein L^(A1 . . . 4),L^(J1 . . . 4), n, n′ . . . q, s and M have the definitions recitedabove.
 12. A compound selected from: a.4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoicacid (2a); b.4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-3-methylbutanoicacid (3a); c.N³,N³,N⁷,N⁷,5,5-Hexamethyl-10-propylidene-5,10-dihydrodibenzo[b,e]siline-3,7-diamine(4a); d.N³,N³,N⁷,N⁷,5,5-Hexamethyl-10-methylene-5,10-dihydrodibenzo[b,e]siline-3,7-diamine(5a); e.3-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)propanoicacid (22); f.4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-2,2-dimethylbutanoicacid (28); g.4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanoicacid (6a); h.4-(3,7-Bis(dimethylamino)-2,8-difluoro-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoicacid (36) i.3-(3,7-Di(azetidin-1-yI)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)propanoicacid (42); j.4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide(44); k.2,5-Dioxopyrrolidin-1-yl-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoate(45); l.N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide(47); m.N-(4-((4R,7R,10S,13S,19S,E)-7-((1H-Indol-2-yl)methyl)-4-(4-hydroxyphenyl)-8,13,15,19-tetramethyl-2,6,9,12-tetraoxo-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl)butyl)-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide(49); n.4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(4-(4-(6-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)butyl)butanamide(51); o.(2αR,4S,4αS,6R,9S,11S,12S,12αR,12βS)-12β-Acetoxy-9-((3-(4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-4α,8,13,13-tetramethyl-5-oxo-2α,3,4,4α,5,6,9,10,11,12,12α,12β-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-β]oxet-12-ylbenzoate (53); p.8-(4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)octanoicacid (55); q.(2αR,4S,4αS,6R,9S,11S,12S,12αR,12βS)-12β-Acetoxy-9-((3-(8-(4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)octanamido)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-4α,8,13,13-tetramethyl-5-oxo-2α,3,4,4α,5,6,9,10,11,12,12α,12β-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-β]oxet-12-ylbenzoate (56); r.4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)butanamide(58); s.4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide(59); t.4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)-N-(21-chloro-8-oxo-3,6,12,15-tetraoxa-9-azahenicosyl)butanamide(64); u.1-(4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3,6,9,12-tetraoxapentadecan-15-amide(65); v.N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamide(66); w.3-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)amino)-2-(4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-3-oxopropane-1-sulfonicacid (69);